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Biolubricant synthesis using immobilised lipase: Process optimisation of trimethylolpropane oleate production
Process Biochemistry 46 (2011) 2225–2231
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Biolubricant synthesis using immobilised lipase: Process optimisation of trimethylolpropane oleate production Cecilia Orellana Åkerman a,∗ , Anna E.V. Hagström a , M. Amin Mollaahmad a , Stefan Karlsson b , Rajni Hatti-Kaul a a b
Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden AAK Sweden AB, Technical Products and Feed, SE-374 82 Karlshamn, Sweden
a r t i c l e
i n f o
Article history: Received 18 February 2011 Received in revised form 24 June 2011 Accepted 8 August 2011 Available online 17 August 2011 Keywords: Polyol esters Immobilised Candida antarctica lipase B Reaction parameters Biocatalyst stability
a b s t r a c t Synthetic esters based on polyols and fatty acids possess suitable technical and ecological properties for applications as biolubricants, and can replace the mineral oil based lubricants in several applications. In this work, the synthesis of trimethylolpropane (TMP) esters with oleic acid using immobilised lipase B from Candida antarctica (Novozym® 435) has been studied. TMP-trioleate has suitable properties for use as hydraulic fluids, especially at extreme temperatures. The effect of different reaction parameters on the reaction efficiency has been evaluated. The study showed that the formation of the triester product was facilitated at high temperature and biocatalyst concentration, as well as stoichiometric amounts of oleic acid and TMP. The product with the highest triester content exhibited the lowest pour point (−42 ◦ C). The stability of the biocatalyst was however limited at high temperature and polyol concentration. Loss of activity during recycling of the biocatalyst at 70 ◦ C was reduced to some extent by washing it with 2-propanol prior to subsequent run. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Synthetic oleochemical esters are regarded as environmentally benign alternatives for mineral oil based lubricants, exhibiting a combination of excellent technical performance with favourable ecological properties [1,2]. They are easily biodegradable and possess low aquatic toxicity. Such esters are thus highly suited as high performance lubricants for different industrial and automotive applications such as hydraulic fluids, metal working fluids, drilling oils, gear oils and lubricants for power saw chains [1,3]. Oleochemical esters are characterised by the presence of one or more ester bonds with alkyl chains that are normally of plant or animal origin. Polyol esters are made of multifunctional synthetic alcohols like trimethylolpropane (TMP), neopentyl glycol or pentaerythritol. They contain at least one quaternary carbon atom that imparts higher chemical stability to the molecules as compared to e.g. the esters of glycerol, in spite of which they are relatively easily biodegradable. Polyol esters are so far the most common group of biolubricants with TMP esters of oleic acid being the most widely applied for hydraulic fluids [4]. Production of trimethylolpropane trioleate on industrial scale is achieved by reaction of TMP with free fatty acids or esters catalysed by a homogeneous or heterogeneous chemical catalyst such as
∗ Corresponding author. Tel.: +46 046 222 4741; fax: +46 046 222 4713. E-mail address: c orellana [email protected] (C.O. Åkerman). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.08.006
acidic resins, acid oxides, and organic ion exchange resins [5]. Synthesis of polyol esters by transesterification with triglycerides using zinc (II) oxalate as catalyst has also been reported [6]. Yet another catalyst used for esterification and transesterification reactions has been the enzyme lipase [7–10]. Earlier studies with immobilised lipases from Rhizomucor miehei and Candida rugosa have shown that the concentration of biocatalyst needed for efficient production was as high as 20–50% (w/v). Recently, the synthesis of TMP-oleate using different heterogeneous catalysts has been compared [11]. The reaction performance using immobilised C. antarctica lipase B, Novozym® 435 (N435), and silica sulphuric acid were comparable, however, the product quality was better with the enzymatically catalysed process. High yields (95%) of the triester were achieved in the process run at 70 ◦ C and the product was found to have suitable properties as biolubricant. In this paper, the effect of different reaction parameters on the efficiency of the synthesis of TMP-oleate using N435 and on enzymatic stability was studied, with an aim to find conditions for an economical and environmentally benign process. 2. Materials and methods 2.1. Materials Oleic acid (OA), 90% (technical grade), was purchased from Alfa Aesar (Karlsruhe, Germany). Trimethylolpropane (TMP) was a kind gift from Perstorp Speciality Chemicals AB (Perstorp, Sweden),
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oleic acid (Tefacid olein, 74% OA) was donated by AAK Sweden AB (Karlshamn, Sweden), while immobilised C. antarctica lipase B, Novozym® 435 (N435) was provided by Novozymes A/S (Bagsvaerd, Denmark). All the solvents used were of analytical grade and were obtained from standard sources.
Initial reaction rate was calculated based on the formation of monoester, within the linear range (up to maximum 15% conversion of OA). Productivity was based on the amount of triester formed after 24 h, divided by the amount of biocatalyst used for each reaction.
2.2. Synthesis of TMP-oleate esters
2.5. Total acid number (TAN)
OA and TMP were mixed at various molar ratios to a total mass of 100 g in a 500 mL reactor. A temperature-controlled oil bath was used to control the reaction temperature. N435 was added once the TMP was dissolved and the reaction mixture was mixed using an overhead stirrer at 400 rpm. Water was removed from the reaction by applying vacuum (20 mbar) using a vacuum pump (LABOPORT® Vacuum Systems) with separator and condenser (KNF-Neuberger AB, Stockholm, Sweden). Samples were withdrawn during the course of the reaction and analysed by titration of the acid groups as well as by HPLC. The initial reaction rate, productivity and substrate conversion were determined under varying reaction conditions. The reaction was stopped by separating the catalyst from the reaction mixture by filtration over a glass filter (40–60 m, Millipore); the catalyst was subsequently washed and the residual enzymatic activity was measured. Synthesis of TMP-oleate was further performed at 1 kg scale using industrial grade oleic acid (90%) and TMP at a ratio of 3.3:1, and 2% (w/w) N435. The reaction was performed in a 2 L batch reactor, the mixture was stirred using an over-head stirrer with a propeller and 20 mbar vacuum was applied. The temperature in the reaction mixture was controlled by a heating mantle connected to a thermometer inserted into the reaction liquid. Samples were collected, analysed and the reaction was stopped as described above.
The reactions were monitored by titration of the acid groups remaining in the sample according to the international standard ISO 6618. Approximately 0.1 g of the sample was dissolved in 10 mL of a solution (containing toluene:2-propanol:water at a ratio of 500:495:5). Thereafter 2 drops of p-naphtholbenzein solution (1 g in 100 mL of the latter solvent) were added. The solution was titrated with 0.1 M potassium hydroxide (KOH) solution in 2-propanol until a shift in colour was observed. All the samples were analysed in triplicates. The total acid number (TAN) was calculated as the quantity of base, expressed in milligrams of potassium hydroxide (KOH) per gram of sample, which is required for titration of the acid groups using Eq. (1).
2.3. Reusability of the biocatalyst A study on determining the reusability of the biocatalyst for the synthesis of TMP-oleate was performed in 100 g scale. Consecutive reactions were performed using 100 g of OA:TMP (molar ratio of 3:1) and 2% (w/w) N435 at 70 ◦ C. After 24 h the reaction liquid was withdrawn (while making sure no biocatalyst was removed) and thereafter new substrates were added. The residual enzymatic activity was calculated by comparison of the initial reaction rate (based on total acid number) for each batch. The initial reaction rate of the first batch was regarded as 100%. The reusability studies with and without 2-propanol treatment were run in 1 g scale using OA:TMP ratio of 3:1 and 2% (w/w) N435 in 4 mL open vials placed in a temperature controlled shaker (MKR 13, HLC Biotech, Germany) at 700 rpm and 70 ◦ C. The residual enzymatic activity (%) in this case was calculated based on the conversion after 24 h for each cycle. The conversion after 24 h using a fresh enzyme was regarded as 100%. 2.4. HPLC analyses Analyses of fatty acids, TMP, and their ester products were performed by gel permeation chromatography (GPC) on two columns of Shodex GPC KF-801 connected in series using a PerkinElmer HPLC system equipped with a refractive index detector L-2490 (Hitachi) with temperature control and an oven (PerkinElmer series 200), both maintained at 35 ◦ C. Tetrahydrofuran was used as eluant at a flow rate of 0.5 mL min−1 . The components were eluted on the basis of the molecular mass in descending order. Typically 10–15 mg of sample from the reactor was diluted in 1 mL HPLC-grade tetrahydrofuran and filtered through a PTFE membrane filter (0.45 m) prior to injection onto the HPLC column.
TAN =
MwKOH × Ctitrant × Vtitrant msample
(1)
where Ctitrant (M), Vtitrant (mL), and MwKOH (g/mol) are KOH concentration, volume and molecular weight, respectively, and msample (g) is the mass of the sample. The acid conversion was then calculated from the total acid number (TAN) of the sample at time t (TANt ) and time zero (TAN0 ), respectively, according to Eq. (2). Conversion =
TAN0 − TANt TAN0
(2)
2.6. Determination of the pour point The pour point is the lowest temperature at which the sample is still poured, while cooled under specific standard conditions, and was measured according to ISO 3016. It was measured using an ISL CPP 5Gs, which automatically determines the pour point using a temperature program. 2.7. Determination of the residual enzymatic activity After the reaction was stopped by filtration, the biocatalyst (N435) was collected and washed first with toluene (25 mL) to remove the oily residue, and thereafter with ethanol (25 mL) and then left to dry in a fume hood. The residual enzymatic activity of the biocatalyst was determined by weighing up ∼30 mg of the dried N435 (in triplicates), to which 3 mL solution of 100 mM caprylic acid in ethanol (90%, v/v) was added. The initial esterification rate was measured at 40 ◦ C during the first 15 min by withdrawal of 100 L samples at intervals, which were immediately diluted with 900 L ethanol and then analysed by gas chromatography. The residual enzymatic activity was calculated by comparing the initial esterification rate for the used N435 with the rate for fresh N435 (washed and dried using the same procedure as described above). 2.8. Gas chromatography GC analyses were performed on a Varian 430 gas chromatograph, equipped with a flash injector, a flame ionisation detector, an autosampler, and software for the data analysis, Galaxy Gas Chromatography. Separation was performed on a NukolTM capillary column (15 m length, 0.53 mm i.d., 0.5 m df ) from Supelco (Sigma–Aldrich, Steinheim, Germany) with a column temperature program from 75 ◦ C to 184 ◦ C. Both the injector and the detector were held at 190 ◦ C and the flow rate of the carrier gas, helium, was
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231
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Table 1 Experimental design for determination of the effect of reaction parameters on esterification reaction between OA and TMP catalysed by N435. Reaction no.
Temperaturea (◦ C)
Ratio OA:TMP
Biocatalyst (%)
Initial reaction rate −1 (mmolmonoester g−1 enz min )
Productivity after 24 h (gtriester /genz )
Conversion OA (%)
Conversion OH (%)
Residual enzymatic activity after 24 h (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
60 100 60 100 60 100 60 100 80 80 80 60 60 70 70 70 100 80 70 70 60 100
1:1 1:1 3:1 3:1 1:1 1:1 3:1 3:1 2:1 2:1 2:1 3:1 3.3:1 3:1 3.3:1 4.5:1 3:1 3:1 2:1 1:1 3.1 3:1
0.5 0.5 0.5 0.5 5 5 5 5 2.75 2.75 2.75 2 2 2 2 2 2 2 2 2 7.5 7.5
0.12 0.38 0.08 0.54 0.07 0.15 0.06 0.28 0.18 0.19 0.21 0.09 0.02 0.11 0.13 0.07 0.20 0.13 0.13 0.12 0.05 0.20
0.0 0.6 9.9 37 1.4 0.7 10 19 15 16 15 4.9 12 11 14 6.4 39 13 13 1.8 2 13
41 56 21 62 99 99 82 96 98 98 99 62 75 73 70 74 92 80 98 99 69 98
14 19 21 62 33 33 82 96 65 66 66 62 75 73 70 74 92 80 65 49 69 98
63 0 89 5 83 2 83 5 52 37 52 6b 76b 87 40b 52 11 40 57 45 – –
Temperature in oil bath. The temperature inside the reactor was approximately 5 ◦ C lower. Measured after 72–76 h.
6 mL min−1 resulting in an analysis time of 7 min. The retention times were 2.3 and 5.5 min for ethyl caprylate and caprylic acid, respectively. 3. Results and discussion In order to characterise the process, solvent-free esterification reactions between oleic acid and TMP were run at 100 g scale under varying experimental conditions. The reactions were run under vacuum based on the small scale tests in 4 mL vials that showed low reaction efficiency when run in closed vials i.e. without water removal as compared to that in open vials which allowed some water evaporation. For example it took about 10 days for the reaction between OA and TMP (at molar ratio of 3:1) at 70 ◦ C to reach equilibrium at 70% conversion of OA in the closed system while 75% conversion was reached within 24 h in the open vials. The reaction parameters that were analysed were the molar ratio of reactants (OA:TMP of 1:1–4.5:1), biocatalyst concentration (0.5–7.5%, w/w) and temperature (60–100 ◦ C). The lowest temperature chosen for the reaction was 60 ◦ C, which is slightly above the melting point of TMP. The result of the reactions; i.e. initial reaction −1 −1 rate (mmolmonoester g−1 enz min ), productivity (gtriester genz ) as well as the percent conversion of acid and hydroxyl-groups and residual enzymatic activity after 24 h are shown in Table 1. Product and residual substrate compositions after 24 h are shown in Table 2. The reaction between OA and TMP occurred to a certain extent without addition of biocatalyst as revealed by 12 and 35% conversion after 24 h at 70 and 100 ◦ C, respectively. 3.1. Effect of reaction temperature As seen for reactions 1–8 in Table 1, an increase in temperature from 60 to 100 ◦ C at a fixed OA:TMP ratio and biocatalyst loading generally resulted in increased initial reaction rate, productivity and substrate conversion. Productivity of (37 gtriester g−1 enz ) was reached in reaction 4 at 100 ◦ C where the biocatalyst amount was limiting (0.5%), however the substrate conversion was only 62% after 24 h. This was attributed to the thermal denaturation of the enzyme, and was confirmed in reaction 8 in which the increase in
biocatalyst concentration to 5% improved the substrate conversion to 96% primarily to triester (Table 2), although the productivity per biocatalyst weight was reduced to half. The intermediate biocatalyst loading (2%) was found to be the optimal one resulting in high productivity (39 gtriester g−1 enz ) and high conversion of substrates (92%) at 100 ◦ C. Reactions 9–11 were run at 80 ◦ C under similar conditions of OA:TMP molar ratio (2:1) and N435 concentration (2.75%, w/w) and showed very similar results with almost quantitative OA conversion and intermediate productivity (∼15 gtriester g−1 enz ). These runs further revealed high reproducibility of the 100 g scale reactions and good control of experimental error. Fig. 1 shows the profile of conversion of acid groups with time during reaction at 60, 70, 80 and 100 ◦ C using stoichiometric amounts of oleic acid and TMP (3:1) and 2% (w/w) N435. This clearly shows an increased reaction rate with increasing temperature. The change in composition of the reaction mixture over time is seen in Fig. 2 for 70 and 100 ◦ C only. The conversion rates −1 of OA at 70 and 100 ◦ C were 0.08, and (0.19 mmol g−1 enz min ), respectively (according to the HPLC data) until the majority of the TMP molecules were converted to the diester form (Fig. 2). Thereafter, esterification of the remaining hydroxyl groups occurred at a slower rate, which is seen as decreased conversion rates of OA; (0.014 and 0.030 mmol g−1 min−1 ) at 70 and 100 ◦ C, respectively 100
Conversion (%)
a b
80 60 40 20 0
0
10
20
30
40
50
60
70
80
Time (h) Fig. 1. Conversion of acid groups during esterification of OA with TMP (molar ratio of 3:1), catalysed by N435 (2%, w/w). () 60 ◦ C, (×) 70 ◦ C, (䊉) 80 ◦ C, and () 100 ◦ C.
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Table 2 Composition of the products and residual substrates obtained after 24 h from the reactions listed in Table 1. Temperaturea (◦ C)
Reaction no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Ratio OA:TMP
60 100 60 100 60 100 60 100 80 80 80 60 60 70 70 70 100 80 70 70 60 100
1:1 1:1 3:1 3:1 1:1 1:1 3:1 3:1 2:1 2:1 2:1 3:1 3.3:1 3:1 3.3:1 4.5:1 3:1 3:1 2:1 1:1 3:1 3:1
Biocatalyst (%, w/w)
0.5 0.5 0.5 0.5 5 5 5 5 2.75 2.75 2.75 2 2 2 2 2 2 2 2 2 7.5 7.5
Products (% w/w)
Substrates (% w/w)
Monoester
Diester
Triester
OA
TMP
24 42 19 8 43 46 n.d. n.d. 13 13 12 4 n.d. n.d. 1 n.d. n.d. n.d. 10 44 3 0
13 14 24 44 38 47 35 6 45 45 44 59 50 57 48 43 18 54 63 43 54 2
n.d. n.d. 5 19 7 3 52 94 42 43 40 10 25 22 27 13 78 27 25 4 17 98
40 29 52 29 2 n.d. 14 n.d. n.d. n.d. 3 27 25 21 24 44 5 20 1 n.d. 25 n.d.
23 15 n.d. n.d. 9 4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8 n.d. n.d.
n.d: not detected. a Temperature in oil bath. The temperature inside the reactor was approximately 5 ◦ C lower.
(Fig. 2). The total reaction time needed to achieve substrate conversion above 95% was around 24 h at 100 ◦ C, 63.5 h at 70 ◦ C, and 94 h at 60 ◦ C. Although a decrease in reaction rate with decreasing concentrations of substrates during the course of the reaction is common, it is more pronounced in this system since the TMP diester is too bulky for interacting with the active site of the enzyme, hence reducing the rate of triester formation and the resultant reaction productivity. Increase in temperature facilitates the reaction due to increased
Concentration (%)
A 100 80
3.2. Effect of biocatalyst loading and initial molar substrate ratio
60
The effect of N435 loading on substrate conversion and productivity at 60 and 100 ◦ C is shown in Fig. 4. The initial reaction rate decreases with increasing biocatalyst load at both temperatures, however the productivities after 24 h show opposite trends. While the productivity increases at 60 ◦ C, it decreases at 100 ◦ C at N435
40 20 0
mobility as well as conformational flexibility of the molecules. On the other hand, the reaction rate at any particular time of the reaction would also depend on the catalytic activity retained by the biocatalyst that is inversely affected by the temperature, especially during the latter part of the reaction. The residual enzymatic activity measured after 24 h of reaction showed that N435 retained over 80% of its original activity when used at 60 and 70 ◦ C, and with further increase in temperature there is a drastic drop in activity to 40% at 80 ◦ C and to 5% at 100 ◦ C (Fig. 3). N435 has long-term stability at 60 ◦ C, according to the supplier. However besides temperature, other factors such as effect of substrates and loss of enzyme from the carrier [12] may also contribute to the loss of activity observed in this work.
0
10
20
30
40
Time (h)
50
60
70
Concentration (%)
B 100 80 60 40 20 0
0
5
10
15
Time (h)
20
25
Fig. 2. Esterification of OA with TMP (molar ratio of 3:1) at (A) 70 ◦ C, and (B) 100 ◦ C, catalysed by 2% (w/w) N435. () OA, (×) TMP, (−) TMP monoester, () TMP diester, and () TMP triester.
Fig. 3. Residual enzymatic activity after 24 h of reaction at different temperatures, OA and TMP were mixed in molar ratio of 3:1, and 2% (w/w) N435 was used, except for 60 ◦ C where 5% N435 was used.
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231
0.4 30
20 0.2 10
0.0
0 0
1
2
3
4
5
6
7
8
Biocatalyst loading (% w/w) Fig. 4. Effect of the biocatalyst (N435) loading on initial reaction rate (open symbols) and productivity of triester after 24 h (closed symbols) using OA:TMP ratio of 3:1 at (䊉/) 60 ◦ C and (/) 100 ◦ C.
concentration higher than 2% (w/w), suggesting that the biocatalyst amount is in excess above this loading. The OA:TMP ratio used for the reaction was varied between 1 and 4.5 and was found to influence mainly the product composition in terms of the relative proportions of TMP-monoester, -diester and -triester (Tables 1 and 2). For obtaining complete esterification of TMP, the minimum OA:TMP ratio should be 3:1. Hence at lower ratios a mixture of mono- and diesters is expected, with their relative amounts being influenced by the other reaction parameters. As seen in Table 1, at low OA:TMP ratios (1–2) the oleic acid was almost completely utilised at all temperatures (60–100 ◦ C) provided the biocatalyst amount was not limiting. This limitation was seen for reactions with 0.5% N435 (reactions 1 and 2 in Table 1) where significant amounts of the substrates remained unutilised and the product obtained had a higher proportion of monoester than the diester irrespective of the temperature (Table 2). With 5% N435 (reactions 5 and 6), equal proportions of mono- and diesters as well as a small amount of triesters were obtained. At OA:TMP ratio of 2:1 (reactions 9–11), di- and triesters were formed in equal proportions along with a lower fraction of monoester (Table 2). Increasing the substrate ratio to 3:1 but keeping the biocatalyst concentration at 0.5% gave no significant change in product profile for 60 ◦ C (reaction 3), while for reaction 4 at 100 ◦ C the product was improved containing diesters as the major product followed by triesters and monoesters, respectively. With 2% N435, only diesters and triesters were formed at all the temperatures, the former predominating at 60–70 ◦ C (reactions 12 and 14) and the latter at 100 ◦ C. With excess of biocatalyst (reactions 7 and 8), triester was the major product especially at 100 ◦ C. At slight excess of oleic acid (OA:TMP of 3.3:1) used in reactions 13 and 15, at 60 and 70 ◦ C, respectively, the reaction productivity was improved. Higher excess (ratio 4.5:1, reaction 16) on the other hand resulted in lower productivity. Fig. 5 shows initial reaction rate and productivity vs. initial molar ratio of OA:TMP at 70 ◦ C. The initial reaction rate is almost constant up to a ratio of 3.3:1 and decreased at higher ratio. This is most probably due to the reduced concentration of OH groups. Also inhibition of the enzyme by the high concentration of acid could be a contributing factor [13,14]. The reaction productivity was the highest at OA:TMP ratio of 3.3:1. Much of the esterified product at 24 h was in the form of diester along with some triester and unutilised OA. The reaction between OA and TMP (ratio 3.3) at 70 ◦ C was also performed at 1 kg scale. No difference compared to the results from 100 g scale could be seen (data not shown). The unreacted excess of fatty acids in the product was removed by distillation (a process called deodorisation), giving no effect on the final product. This means that if a slight excess of OA is used in the process, it can be
0.15
30
0.10
20
0.05
10
0.00
Productivity, 24 h (gtriester*genz-1)
40
Initial rea action rate (mmolmonoo*genz-1*min-1)
50
Productivity, 24 h (gtriester*genz-1)
Initial reaction rate (mmolmonoester*genz-1*min-1)
0.6
2229
0 0
1
2
3
4
5
Molar Ratio (OA:TMP) Fig. 5. Initial reaction rate () and productivity of triester after 24 h (䊉), in relation to the initial ratio of OA:TMP at 70 ◦ C, using 2% N435.
removed in order to improve the product quality, such as lower acid value as well as lower pour point. Samples of products obtained from some of the reactions were selected for pour point analysis. The pour point values were −27, −42 and −30 ◦ C for the samples from reactions 7, 8 and 11 respectively. A pour point below −40 ◦ C is the desired value for biolubricants to be used as hydraulic fluids in cold regions, and was obtained for the sample containing mainly TMP trioleate. Samples 7 and 11 had a relatively high amount of diester, and additionally even unutilised substrates were present in sample 7, which is reflected in the higher pour point values. This shows the importance of high conversions in order to fulfil the product requirements for hydraulic fluids. 3.3. Biocatalyst stability Residual enzymatic activity after incubation at 70 ◦ C with substrates (OA and TMP) and product (triester), respectively, as well as after the reactions were investigated in order to know more about the factors underlying the loss of biocatalyst activity. Both the substrates and the products themselves reduced the enzymatic activity, TMP being the most aggressive one. The deactivating effect of TMP was confirmed by decreasing residual activity with increasing amount of the polyol in the reaction (Table 1), and is likely to be due to the detrimental effect of the neopentyl group of TMP on the lipase as suggested earlier [8]. Further investigations would be needed to determine if this effect is due to enzyme denaturation and/or leakage from the carrier. Fig. 6 shows N435 recycled for consecutive esterification reactions run for 24 h each, at 70 ◦ C at 100 g scale. There was a uniform decrease of activity after each cycle resulting in 20% retention after the sixth cycle and a half-life of 94 h, which is lower than some [15,16], but higher than other [17] previously determined half-lives for this biocatalyst. The rapid decrease in the enzyme activity could be due to the residual polyol remaining on the biocatalyst and/or leakage from the carrier [12]. The possibility to improve the biocatalyst performance by washing with 2-propanol in between runs was investigated in 1 g scale. As seen in Fig. 6 (insert), in contrast to the recycling of N435 without any intermediate treatment, washing the enzyme beads with 2-propanol resulted in no loss in activity during the first 3 runs but thereafter almost 20% of the activity was lost after each run. It is possible that 2-propanol helps to remove the residual polyol from the biocatalyst beads, and the biocatalyst half-life was almost doubled as compared to when the biocatalyst was reused without washing with 2-propanol. Although washing the biocatalyst is shown to have a positive effect on the stability, the stability is still low, and poses the main limitation in its application as a catalyst for this reaction.
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231 Residual enzymatic activity (%)
2230
100
Residual enzymatic activity (%)
90 80
120 100 80 60 40 20 0 0
24
48
72
96
120
144
Time (h)
70 60 50 40 30 20 10 0 0
24
48
72
96
120
144
Time (h) Fig. 6. Residual enzymatic activity, determined by calculation of initial reaction rate based on acid titration, for consecutive esterification reactions at 70 ◦ C, 2% (w/w) N435 and OA:TMP molar ratio of 3:1 at 100 g scale. The graph inside shows the residual enzymatic activity, without washing (black) and with washing using 2-propanol each 24 h (grey), at the same reaction conditions as above in 1 mL scale. The activity was calculated based on conversion of OA after 24 h reaction (TAN).
3.4. Concluding remarks It was shown in our earlier study that esterification of the triol, TMP with oleic acid catalysed by N435 had a lower environmental impact and also yielded a product with good biolubricant properties in comparison to that obtained with other heterogeneous catalysts [11]. This study analyses the effect of the process variables on the reaction efficiency and shows that high temperature and high biocatalyst loading as well as a stoichiometric excess of oleic acid are favourable for the reaction. Lipase catalysed synthesis of TMP-trioleate is expected to involve OA as the acyl donor (OA) and a number of acyl acceptors, i.e. TMP, TMP-monoester and TMPdiester, as the reaction progresses. These polyol esters may also function as acyl donors, and water acts as the acyl acceptor under the conditions of incomplete water removal that would result in hydrolysis of the esters. C. antarctica lipase B, the enzyme used in this study, has a large acyl donor site but a narrow acyl acceptor site [18], that may restrict the binding of the TMP-ester intermediates. The reaction was thus observed to take place with relatively high efficiency until the formation of diester product and is thereafter affected probably due to the bulkiness of the diester. The high temperature required for the reaction and also the polyol seem to have a detrimental effect on the enzyme activity, resulting in limited biocatalyst reusability and economic feasibility. Considering the low market price for speciality chemicals like biolubricants (in the range of 1 Euro/kg) and high cost of N435 (900 Euros/kg) at least 30-fold improvement in the biocatalyst activity/stability is estimated to be necessary to reach an economical process. Different strategies may be used to reduce the cost and improve the stability of the lipase. It has earlier been shown in our laboratory that the choice of a suitable matrix and optimising the amount of enzyme immobilised on the matrix can significantly influence the biocatalyst cost [19]. It was also suggested that increasing the scale of the process could drastically reduce the production cost of the biocatalyst [19]. Improvements can also be made by choosing or developing an enzyme with optimal molecular features such as higher stability to temperature and the polyol. For example, C. antarctica lipase B variant with enhanced thermostability at 90 ◦ C has been obtained
by random mutagenesis using an error prone PCR approach [20]. Another approach being investigated by us is to widen the acyl acceptor site so as to accommodate the TMP mono- and diesters and hence lead to a shorter reaction time and thereby less exposure of the enzyme to the harsh process conditions. Acknowledgments This work was performed within the framework of Greenchem – Speciality Chemicals from renewable resources, a research programme at Lund University, Sweden, supported by the Foundation for Strategic Environmental Research (Mistra). Roger Jacobsson, Jerker Johansson and Yvonne Samuelsson at AAK Sweden AB (Karlshamn, Sweden) are gratefully acknowledged for valuable discussions as well as for their help with analyses, product refining and evaluation. References [1] Willing A. Lubricants based on renewable resources – an environmentally compatible alternative to mineral oil products. Chemosphere 2001;43:89–98. [2] Schneider MP. Plant-oil-based lubricants and hydraulic fluids. J Sci Food Agric 2006;86:1769–80. [3] Ridderikhoff H, Oosterman J. Biodegradable hydraulic fluids: rheological behaviour at low temperatures of several oleochemically derived synthetic esters. Synth Lubr 2005;21:299–313. [4] Mang T, Dresel W. Lubricants and lubrication. Weinheim; 2001. [5] Fuchs Petrolub A-G. Synthetic lubricants and hydraulic fluids prepared by electrophilic addition of carboxylic acids to unsaturated fatty acids or esters. Germany DE Patent WO 2001053438; 2001. [6] Henkel K.-G.a.A., Transesterification method for the preparation of fatty acid polyol esters from triglycerides and polyhydric alcohols. Germany DE Patent WO 9957092; 1998. [7] Linko YY, Tervakangas T, Lamsa M, Linko P. Production of trimethylolpropane esters of rapeseed oil fatty acids by immobilized lipase. Biotechnol Tech 1997;11:889–92. [8] Monot F, Benoit Y, Vallerini D, Vandecasteele JP. Enzymatic synthesis of neopentylpolyol esters in organic media. Appl Biochem Biotechnol 1990;24–25:375–86. [9] Uosukainen E, Linko Y-Y, Lamsa M, Tervakangas T, Linko P. Transesterification of trimethylolpropane and rapeseed oil methyl ester to environmentally acceptable lubricants. J Am Oil Chem Soc 1998;75:1557–63. [10] People Republic of China. Process for synthesizing polyol ester with lipase catalyst. CN Patent CN 101475467; 2009.
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231 [11] Åkerman CO, Gaber Y, Abd Ghanid N, Lämsäe M, Hatti-Kaul R. Clean synthesis of biolubricants for low temperature applications using heterogeneous catalysts. J Mol Catal B Enzym 2011;72:263–9. [12] Chen B, Hu J, Xie W, Cai M, Gross R. Candida antarctica lipase B chemically immobilized on epoxy-activated Micro and nanobeads: catalysts for polyesters synthesis. Biomacromolecules 2008;9:463–71. [13] Goderis HL, Ampe G, Feyten MP, Fouwe BL, Guffens WM, Van Cauwenbergh SM, et al. Lipase-catalyzed ester exchange reactions in organic media with controlled humidity. Biotechnol Bioeng 1987;30:258–66. [14] Garcia T, Coteron A, Martinez M, Aracil J. Kinetic model for the esterification of oleic acid and cetyl alcohol using an immobilized lipase as catalyst. Chem Eng Sci 2000;55:1411–23. [15] Watanabe Y, Pinsirodom P, Nagao T, Yamauchi A, Kobayashi T, Nishida Y, et al. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase. J Mol Catal B Enzym 2007;44:99–105.
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[16] Hagström AEV, Nordblad M, Adlercreutz P. Biocatalytic polyester acrylationprocess optimization and enzyme stability. Biotechnol Bioeng 2009;102: 693–9. [17] Hilterhaus L, Thum O, Liese A. Reactor concept for lipase-catalyzed solvent-free conversion of highly viscous reactants forming two-phase systems. Org Process Res Dev 2008;12:618–25. [18] Naik S, Basu A, Saikia R, Madan B, Paul P, Chaterjee R, et al. Lipases for use in industrial biocatalysis: specificity of selected structural groups of lipases. J Mol Catal B Enzym 2010;65:18–23. [19] Tufvesson P, Törnvall U, Carvalho J, Karlsson A, Hatti-Kaul R. Towards a costeffective immobilized lipase for the synthesis of specialty chemicals. J Mol Catal B Enzym 2011;68:200–5. [20] Chodorge M, Fourage L, Ullmann C, Duvivier V, Masson JM, Lefevre F. Rational strategies for directed evolution of biocatalysts: application to Candida antarctica lipase B (CALB). Adv Synth Catal 2005;347:1022–6.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biolubricant synthesis using immobilised lipase: Process optimisation of trimethylolpropane oleate production Cecilia Orellana Åkerman a,∗ , Anna E.V. Hagström a , M. Amin Mollaahmad a , Stefan Karlsson b , Rajni Hatti-Kaul a a b
Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden AAK Sweden AB, Technical Products and Feed, SE-374 82 Karlshamn, Sweden
a r t i c l e
i n f o
Article history: Received 18 February 2011 Received in revised form 24 June 2011 Accepted 8 August 2011 Available online 17 August 2011 Keywords: Polyol esters Immobilised Candida antarctica lipase B Reaction parameters Biocatalyst stability
a b s t r a c t Synthetic esters based on polyols and fatty acids possess suitable technical and ecological properties for applications as biolubricants, and can replace the mineral oil based lubricants in several applications. In this work, the synthesis of trimethylolpropane (TMP) esters with oleic acid using immobilised lipase B from Candida antarctica (Novozym® 435) has been studied. TMP-trioleate has suitable properties for use as hydraulic fluids, especially at extreme temperatures. The effect of different reaction parameters on the reaction efficiency has been evaluated. The study showed that the formation of the triester product was facilitated at high temperature and biocatalyst concentration, as well as stoichiometric amounts of oleic acid and TMP. The product with the highest triester content exhibited the lowest pour point (−42 ◦ C). The stability of the biocatalyst was however limited at high temperature and polyol concentration. Loss of activity during recycling of the biocatalyst at 70 ◦ C was reduced to some extent by washing it with 2-propanol prior to subsequent run. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Synthetic oleochemical esters are regarded as environmentally benign alternatives for mineral oil based lubricants, exhibiting a combination of excellent technical performance with favourable ecological properties [1,2]. They are easily biodegradable and possess low aquatic toxicity. Such esters are thus highly suited as high performance lubricants for different industrial and automotive applications such as hydraulic fluids, metal working fluids, drilling oils, gear oils and lubricants for power saw chains [1,3]. Oleochemical esters are characterised by the presence of one or more ester bonds with alkyl chains that are normally of plant or animal origin. Polyol esters are made of multifunctional synthetic alcohols like trimethylolpropane (TMP), neopentyl glycol or pentaerythritol. They contain at least one quaternary carbon atom that imparts higher chemical stability to the molecules as compared to e.g. the esters of glycerol, in spite of which they are relatively easily biodegradable. Polyol esters are so far the most common group of biolubricants with TMP esters of oleic acid being the most widely applied for hydraulic fluids [4]. Production of trimethylolpropane trioleate on industrial scale is achieved by reaction of TMP with free fatty acids or esters catalysed by a homogeneous or heterogeneous chemical catalyst such as
∗ Corresponding author. Tel.: +46 046 222 4741; fax: +46 046 222 4713. E-mail address: c orellana [email protected] (C.O. Åkerman). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.08.006
acidic resins, acid oxides, and organic ion exchange resins [5]. Synthesis of polyol esters by transesterification with triglycerides using zinc (II) oxalate as catalyst has also been reported [6]. Yet another catalyst used for esterification and transesterification reactions has been the enzyme lipase [7–10]. Earlier studies with immobilised lipases from Rhizomucor miehei and Candida rugosa have shown that the concentration of biocatalyst needed for efficient production was as high as 20–50% (w/v). Recently, the synthesis of TMP-oleate using different heterogeneous catalysts has been compared [11]. The reaction performance using immobilised C. antarctica lipase B, Novozym® 435 (N435), and silica sulphuric acid were comparable, however, the product quality was better with the enzymatically catalysed process. High yields (95%) of the triester were achieved in the process run at 70 ◦ C and the product was found to have suitable properties as biolubricant. In this paper, the effect of different reaction parameters on the efficiency of the synthesis of TMP-oleate using N435 and on enzymatic stability was studied, with an aim to find conditions for an economical and environmentally benign process. 2. Materials and methods 2.1. Materials Oleic acid (OA), 90% (technical grade), was purchased from Alfa Aesar (Karlsruhe, Germany). Trimethylolpropane (TMP) was a kind gift from Perstorp Speciality Chemicals AB (Perstorp, Sweden),
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oleic acid (Tefacid olein, 74% OA) was donated by AAK Sweden AB (Karlshamn, Sweden), while immobilised C. antarctica lipase B, Novozym® 435 (N435) was provided by Novozymes A/S (Bagsvaerd, Denmark). All the solvents used were of analytical grade and were obtained from standard sources.
Initial reaction rate was calculated based on the formation of monoester, within the linear range (up to maximum 15% conversion of OA). Productivity was based on the amount of triester formed after 24 h, divided by the amount of biocatalyst used for each reaction.
2.2. Synthesis of TMP-oleate esters
2.5. Total acid number (TAN)
OA and TMP were mixed at various molar ratios to a total mass of 100 g in a 500 mL reactor. A temperature-controlled oil bath was used to control the reaction temperature. N435 was added once the TMP was dissolved and the reaction mixture was mixed using an overhead stirrer at 400 rpm. Water was removed from the reaction by applying vacuum (20 mbar) using a vacuum pump (LABOPORT® Vacuum Systems) with separator and condenser (KNF-Neuberger AB, Stockholm, Sweden). Samples were withdrawn during the course of the reaction and analysed by titration of the acid groups as well as by HPLC. The initial reaction rate, productivity and substrate conversion were determined under varying reaction conditions. The reaction was stopped by separating the catalyst from the reaction mixture by filtration over a glass filter (40–60 m, Millipore); the catalyst was subsequently washed and the residual enzymatic activity was measured. Synthesis of TMP-oleate was further performed at 1 kg scale using industrial grade oleic acid (90%) and TMP at a ratio of 3.3:1, and 2% (w/w) N435. The reaction was performed in a 2 L batch reactor, the mixture was stirred using an over-head stirrer with a propeller and 20 mbar vacuum was applied. The temperature in the reaction mixture was controlled by a heating mantle connected to a thermometer inserted into the reaction liquid. Samples were collected, analysed and the reaction was stopped as described above.
The reactions were monitored by titration of the acid groups remaining in the sample according to the international standard ISO 6618. Approximately 0.1 g of the sample was dissolved in 10 mL of a solution (containing toluene:2-propanol:water at a ratio of 500:495:5). Thereafter 2 drops of p-naphtholbenzein solution (1 g in 100 mL of the latter solvent) were added. The solution was titrated with 0.1 M potassium hydroxide (KOH) solution in 2-propanol until a shift in colour was observed. All the samples were analysed in triplicates. The total acid number (TAN) was calculated as the quantity of base, expressed in milligrams of potassium hydroxide (KOH) per gram of sample, which is required for titration of the acid groups using Eq. (1).
2.3. Reusability of the biocatalyst A study on determining the reusability of the biocatalyst for the synthesis of TMP-oleate was performed in 100 g scale. Consecutive reactions were performed using 100 g of OA:TMP (molar ratio of 3:1) and 2% (w/w) N435 at 70 ◦ C. After 24 h the reaction liquid was withdrawn (while making sure no biocatalyst was removed) and thereafter new substrates were added. The residual enzymatic activity was calculated by comparison of the initial reaction rate (based on total acid number) for each batch. The initial reaction rate of the first batch was regarded as 100%. The reusability studies with and without 2-propanol treatment were run in 1 g scale using OA:TMP ratio of 3:1 and 2% (w/w) N435 in 4 mL open vials placed in a temperature controlled shaker (MKR 13, HLC Biotech, Germany) at 700 rpm and 70 ◦ C. The residual enzymatic activity (%) in this case was calculated based on the conversion after 24 h for each cycle. The conversion after 24 h using a fresh enzyme was regarded as 100%. 2.4. HPLC analyses Analyses of fatty acids, TMP, and their ester products were performed by gel permeation chromatography (GPC) on two columns of Shodex GPC KF-801 connected in series using a PerkinElmer HPLC system equipped with a refractive index detector L-2490 (Hitachi) with temperature control and an oven (PerkinElmer series 200), both maintained at 35 ◦ C. Tetrahydrofuran was used as eluant at a flow rate of 0.5 mL min−1 . The components were eluted on the basis of the molecular mass in descending order. Typically 10–15 mg of sample from the reactor was diluted in 1 mL HPLC-grade tetrahydrofuran and filtered through a PTFE membrane filter (0.45 m) prior to injection onto the HPLC column.
TAN =
MwKOH × Ctitrant × Vtitrant msample
(1)
where Ctitrant (M), Vtitrant (mL), and MwKOH (g/mol) are KOH concentration, volume and molecular weight, respectively, and msample (g) is the mass of the sample. The acid conversion was then calculated from the total acid number (TAN) of the sample at time t (TANt ) and time zero (TAN0 ), respectively, according to Eq. (2). Conversion =
TAN0 − TANt TAN0
(2)
2.6. Determination of the pour point The pour point is the lowest temperature at which the sample is still poured, while cooled under specific standard conditions, and was measured according to ISO 3016. It was measured using an ISL CPP 5Gs, which automatically determines the pour point using a temperature program. 2.7. Determination of the residual enzymatic activity After the reaction was stopped by filtration, the biocatalyst (N435) was collected and washed first with toluene (25 mL) to remove the oily residue, and thereafter with ethanol (25 mL) and then left to dry in a fume hood. The residual enzymatic activity of the biocatalyst was determined by weighing up ∼30 mg of the dried N435 (in triplicates), to which 3 mL solution of 100 mM caprylic acid in ethanol (90%, v/v) was added. The initial esterification rate was measured at 40 ◦ C during the first 15 min by withdrawal of 100 L samples at intervals, which were immediately diluted with 900 L ethanol and then analysed by gas chromatography. The residual enzymatic activity was calculated by comparing the initial esterification rate for the used N435 with the rate for fresh N435 (washed and dried using the same procedure as described above). 2.8. Gas chromatography GC analyses were performed on a Varian 430 gas chromatograph, equipped with a flash injector, a flame ionisation detector, an autosampler, and software for the data analysis, Galaxy Gas Chromatography. Separation was performed on a NukolTM capillary column (15 m length, 0.53 mm i.d., 0.5 m df ) from Supelco (Sigma–Aldrich, Steinheim, Germany) with a column temperature program from 75 ◦ C to 184 ◦ C. Both the injector and the detector were held at 190 ◦ C and the flow rate of the carrier gas, helium, was
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Table 1 Experimental design for determination of the effect of reaction parameters on esterification reaction between OA and TMP catalysed by N435. Reaction no.
Temperaturea (◦ C)
Ratio OA:TMP
Biocatalyst (%)
Initial reaction rate −1 (mmolmonoester g−1 enz min )
Productivity after 24 h (gtriester /genz )
Conversion OA (%)
Conversion OH (%)
Residual enzymatic activity after 24 h (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
60 100 60 100 60 100 60 100 80 80 80 60 60 70 70 70 100 80 70 70 60 100
1:1 1:1 3:1 3:1 1:1 1:1 3:1 3:1 2:1 2:1 2:1 3:1 3.3:1 3:1 3.3:1 4.5:1 3:1 3:1 2:1 1:1 3.1 3:1
0.5 0.5 0.5 0.5 5 5 5 5 2.75 2.75 2.75 2 2 2 2 2 2 2 2 2 7.5 7.5
0.12 0.38 0.08 0.54 0.07 0.15 0.06 0.28 0.18 0.19 0.21 0.09 0.02 0.11 0.13 0.07 0.20 0.13 0.13 0.12 0.05 0.20
0.0 0.6 9.9 37 1.4 0.7 10 19 15 16 15 4.9 12 11 14 6.4 39 13 13 1.8 2 13
41 56 21 62 99 99 82 96 98 98 99 62 75 73 70 74 92 80 98 99 69 98
14 19 21 62 33 33 82 96 65 66 66 62 75 73 70 74 92 80 65 49 69 98
63 0 89 5 83 2 83 5 52 37 52 6b 76b 87 40b 52 11 40 57 45 – –
Temperature in oil bath. The temperature inside the reactor was approximately 5 ◦ C lower. Measured after 72–76 h.
6 mL min−1 resulting in an analysis time of 7 min. The retention times were 2.3 and 5.5 min for ethyl caprylate and caprylic acid, respectively. 3. Results and discussion In order to characterise the process, solvent-free esterification reactions between oleic acid and TMP were run at 100 g scale under varying experimental conditions. The reactions were run under vacuum based on the small scale tests in 4 mL vials that showed low reaction efficiency when run in closed vials i.e. without water removal as compared to that in open vials which allowed some water evaporation. For example it took about 10 days for the reaction between OA and TMP (at molar ratio of 3:1) at 70 ◦ C to reach equilibrium at 70% conversion of OA in the closed system while 75% conversion was reached within 24 h in the open vials. The reaction parameters that were analysed were the molar ratio of reactants (OA:TMP of 1:1–4.5:1), biocatalyst concentration (0.5–7.5%, w/w) and temperature (60–100 ◦ C). The lowest temperature chosen for the reaction was 60 ◦ C, which is slightly above the melting point of TMP. The result of the reactions; i.e. initial reaction −1 −1 rate (mmolmonoester g−1 enz min ), productivity (gtriester genz ) as well as the percent conversion of acid and hydroxyl-groups and residual enzymatic activity after 24 h are shown in Table 1. Product and residual substrate compositions after 24 h are shown in Table 2. The reaction between OA and TMP occurred to a certain extent without addition of biocatalyst as revealed by 12 and 35% conversion after 24 h at 70 and 100 ◦ C, respectively. 3.1. Effect of reaction temperature As seen for reactions 1–8 in Table 1, an increase in temperature from 60 to 100 ◦ C at a fixed OA:TMP ratio and biocatalyst loading generally resulted in increased initial reaction rate, productivity and substrate conversion. Productivity of (37 gtriester g−1 enz ) was reached in reaction 4 at 100 ◦ C where the biocatalyst amount was limiting (0.5%), however the substrate conversion was only 62% after 24 h. This was attributed to the thermal denaturation of the enzyme, and was confirmed in reaction 8 in which the increase in
biocatalyst concentration to 5% improved the substrate conversion to 96% primarily to triester (Table 2), although the productivity per biocatalyst weight was reduced to half. The intermediate biocatalyst loading (2%) was found to be the optimal one resulting in high productivity (39 gtriester g−1 enz ) and high conversion of substrates (92%) at 100 ◦ C. Reactions 9–11 were run at 80 ◦ C under similar conditions of OA:TMP molar ratio (2:1) and N435 concentration (2.75%, w/w) and showed very similar results with almost quantitative OA conversion and intermediate productivity (∼15 gtriester g−1 enz ). These runs further revealed high reproducibility of the 100 g scale reactions and good control of experimental error. Fig. 1 shows the profile of conversion of acid groups with time during reaction at 60, 70, 80 and 100 ◦ C using stoichiometric amounts of oleic acid and TMP (3:1) and 2% (w/w) N435. This clearly shows an increased reaction rate with increasing temperature. The change in composition of the reaction mixture over time is seen in Fig. 2 for 70 and 100 ◦ C only. The conversion rates −1 of OA at 70 and 100 ◦ C were 0.08, and (0.19 mmol g−1 enz min ), respectively (according to the HPLC data) until the majority of the TMP molecules were converted to the diester form (Fig. 2). Thereafter, esterification of the remaining hydroxyl groups occurred at a slower rate, which is seen as decreased conversion rates of OA; (0.014 and 0.030 mmol g−1 min−1 ) at 70 and 100 ◦ C, respectively 100
Conversion (%)
a b
80 60 40 20 0
0
10
20
30
40
50
60
70
80
Time (h) Fig. 1. Conversion of acid groups during esterification of OA with TMP (molar ratio of 3:1), catalysed by N435 (2%, w/w). () 60 ◦ C, (×) 70 ◦ C, (䊉) 80 ◦ C, and () 100 ◦ C.
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Table 2 Composition of the products and residual substrates obtained after 24 h from the reactions listed in Table 1. Temperaturea (◦ C)
Reaction no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Ratio OA:TMP
60 100 60 100 60 100 60 100 80 80 80 60 60 70 70 70 100 80 70 70 60 100
1:1 1:1 3:1 3:1 1:1 1:1 3:1 3:1 2:1 2:1 2:1 3:1 3.3:1 3:1 3.3:1 4.5:1 3:1 3:1 2:1 1:1 3:1 3:1
Biocatalyst (%, w/w)
0.5 0.5 0.5 0.5 5 5 5 5 2.75 2.75 2.75 2 2 2 2 2 2 2 2 2 7.5 7.5
Products (% w/w)
Substrates (% w/w)
Monoester
Diester
Triester
OA
TMP
24 42 19 8 43 46 n.d. n.d. 13 13 12 4 n.d. n.d. 1 n.d. n.d. n.d. 10 44 3 0
13 14 24 44 38 47 35 6 45 45 44 59 50 57 48 43 18 54 63 43 54 2
n.d. n.d. 5 19 7 3 52 94 42 43 40 10 25 22 27 13 78 27 25 4 17 98
40 29 52 29 2 n.d. 14 n.d. n.d. n.d. 3 27 25 21 24 44 5 20 1 n.d. 25 n.d.
23 15 n.d. n.d. 9 4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8 n.d. n.d.
n.d: not detected. a Temperature in oil bath. The temperature inside the reactor was approximately 5 ◦ C lower.
(Fig. 2). The total reaction time needed to achieve substrate conversion above 95% was around 24 h at 100 ◦ C, 63.5 h at 70 ◦ C, and 94 h at 60 ◦ C. Although a decrease in reaction rate with decreasing concentrations of substrates during the course of the reaction is common, it is more pronounced in this system since the TMP diester is too bulky for interacting with the active site of the enzyme, hence reducing the rate of triester formation and the resultant reaction productivity. Increase in temperature facilitates the reaction due to increased
Concentration (%)
A 100 80
3.2. Effect of biocatalyst loading and initial molar substrate ratio
60
The effect of N435 loading on substrate conversion and productivity at 60 and 100 ◦ C is shown in Fig. 4. The initial reaction rate decreases with increasing biocatalyst load at both temperatures, however the productivities after 24 h show opposite trends. While the productivity increases at 60 ◦ C, it decreases at 100 ◦ C at N435
40 20 0
mobility as well as conformational flexibility of the molecules. On the other hand, the reaction rate at any particular time of the reaction would also depend on the catalytic activity retained by the biocatalyst that is inversely affected by the temperature, especially during the latter part of the reaction. The residual enzymatic activity measured after 24 h of reaction showed that N435 retained over 80% of its original activity when used at 60 and 70 ◦ C, and with further increase in temperature there is a drastic drop in activity to 40% at 80 ◦ C and to 5% at 100 ◦ C (Fig. 3). N435 has long-term stability at 60 ◦ C, according to the supplier. However besides temperature, other factors such as effect of substrates and loss of enzyme from the carrier [12] may also contribute to the loss of activity observed in this work.
0
10
20
30
40
Time (h)
50
60
70
Concentration (%)
B 100 80 60 40 20 0
0
5
10
15
Time (h)
20
25
Fig. 2. Esterification of OA with TMP (molar ratio of 3:1) at (A) 70 ◦ C, and (B) 100 ◦ C, catalysed by 2% (w/w) N435. () OA, (×) TMP, (−) TMP monoester, () TMP diester, and () TMP triester.
Fig. 3. Residual enzymatic activity after 24 h of reaction at different temperatures, OA and TMP were mixed in molar ratio of 3:1, and 2% (w/w) N435 was used, except for 60 ◦ C where 5% N435 was used.
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0.4 30
20 0.2 10
0.0
0 0
1
2
3
4
5
6
7
8
Biocatalyst loading (% w/w) Fig. 4. Effect of the biocatalyst (N435) loading on initial reaction rate (open symbols) and productivity of triester after 24 h (closed symbols) using OA:TMP ratio of 3:1 at (䊉/) 60 ◦ C and (/) 100 ◦ C.
concentration higher than 2% (w/w), suggesting that the biocatalyst amount is in excess above this loading. The OA:TMP ratio used for the reaction was varied between 1 and 4.5 and was found to influence mainly the product composition in terms of the relative proportions of TMP-monoester, -diester and -triester (Tables 1 and 2). For obtaining complete esterification of TMP, the minimum OA:TMP ratio should be 3:1. Hence at lower ratios a mixture of mono- and diesters is expected, with their relative amounts being influenced by the other reaction parameters. As seen in Table 1, at low OA:TMP ratios (1–2) the oleic acid was almost completely utilised at all temperatures (60–100 ◦ C) provided the biocatalyst amount was not limiting. This limitation was seen for reactions with 0.5% N435 (reactions 1 and 2 in Table 1) where significant amounts of the substrates remained unutilised and the product obtained had a higher proportion of monoester than the diester irrespective of the temperature (Table 2). With 5% N435 (reactions 5 and 6), equal proportions of mono- and diesters as well as a small amount of triesters were obtained. At OA:TMP ratio of 2:1 (reactions 9–11), di- and triesters were formed in equal proportions along with a lower fraction of monoester (Table 2). Increasing the substrate ratio to 3:1 but keeping the biocatalyst concentration at 0.5% gave no significant change in product profile for 60 ◦ C (reaction 3), while for reaction 4 at 100 ◦ C the product was improved containing diesters as the major product followed by triesters and monoesters, respectively. With 2% N435, only diesters and triesters were formed at all the temperatures, the former predominating at 60–70 ◦ C (reactions 12 and 14) and the latter at 100 ◦ C. With excess of biocatalyst (reactions 7 and 8), triester was the major product especially at 100 ◦ C. At slight excess of oleic acid (OA:TMP of 3.3:1) used in reactions 13 and 15, at 60 and 70 ◦ C, respectively, the reaction productivity was improved. Higher excess (ratio 4.5:1, reaction 16) on the other hand resulted in lower productivity. Fig. 5 shows initial reaction rate and productivity vs. initial molar ratio of OA:TMP at 70 ◦ C. The initial reaction rate is almost constant up to a ratio of 3.3:1 and decreased at higher ratio. This is most probably due to the reduced concentration of OH groups. Also inhibition of the enzyme by the high concentration of acid could be a contributing factor [13,14]. The reaction productivity was the highest at OA:TMP ratio of 3.3:1. Much of the esterified product at 24 h was in the form of diester along with some triester and unutilised OA. The reaction between OA and TMP (ratio 3.3) at 70 ◦ C was also performed at 1 kg scale. No difference compared to the results from 100 g scale could be seen (data not shown). The unreacted excess of fatty acids in the product was removed by distillation (a process called deodorisation), giving no effect on the final product. This means that if a slight excess of OA is used in the process, it can be
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Molar Ratio (OA:TMP) Fig. 5. Initial reaction rate () and productivity of triester after 24 h (䊉), in relation to the initial ratio of OA:TMP at 70 ◦ C, using 2% N435.
removed in order to improve the product quality, such as lower acid value as well as lower pour point. Samples of products obtained from some of the reactions were selected for pour point analysis. The pour point values were −27, −42 and −30 ◦ C for the samples from reactions 7, 8 and 11 respectively. A pour point below −40 ◦ C is the desired value for biolubricants to be used as hydraulic fluids in cold regions, and was obtained for the sample containing mainly TMP trioleate. Samples 7 and 11 had a relatively high amount of diester, and additionally even unutilised substrates were present in sample 7, which is reflected in the higher pour point values. This shows the importance of high conversions in order to fulfil the product requirements for hydraulic fluids. 3.3. Biocatalyst stability Residual enzymatic activity after incubation at 70 ◦ C with substrates (OA and TMP) and product (triester), respectively, as well as after the reactions were investigated in order to know more about the factors underlying the loss of biocatalyst activity. Both the substrates and the products themselves reduced the enzymatic activity, TMP being the most aggressive one. The deactivating effect of TMP was confirmed by decreasing residual activity with increasing amount of the polyol in the reaction (Table 1), and is likely to be due to the detrimental effect of the neopentyl group of TMP on the lipase as suggested earlier [8]. Further investigations would be needed to determine if this effect is due to enzyme denaturation and/or leakage from the carrier. Fig. 6 shows N435 recycled for consecutive esterification reactions run for 24 h each, at 70 ◦ C at 100 g scale. There was a uniform decrease of activity after each cycle resulting in 20% retention after the sixth cycle and a half-life of 94 h, which is lower than some [15,16], but higher than other [17] previously determined half-lives for this biocatalyst. The rapid decrease in the enzyme activity could be due to the residual polyol remaining on the biocatalyst and/or leakage from the carrier [12]. The possibility to improve the biocatalyst performance by washing with 2-propanol in between runs was investigated in 1 g scale. As seen in Fig. 6 (insert), in contrast to the recycling of N435 without any intermediate treatment, washing the enzyme beads with 2-propanol resulted in no loss in activity during the first 3 runs but thereafter almost 20% of the activity was lost after each run. It is possible that 2-propanol helps to remove the residual polyol from the biocatalyst beads, and the biocatalyst half-life was almost doubled as compared to when the biocatalyst was reused without washing with 2-propanol. Although washing the biocatalyst is shown to have a positive effect on the stability, the stability is still low, and poses the main limitation in its application as a catalyst for this reaction.
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231 Residual enzymatic activity (%)
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Time (h) Fig. 6. Residual enzymatic activity, determined by calculation of initial reaction rate based on acid titration, for consecutive esterification reactions at 70 ◦ C, 2% (w/w) N435 and OA:TMP molar ratio of 3:1 at 100 g scale. The graph inside shows the residual enzymatic activity, without washing (black) and with washing using 2-propanol each 24 h (grey), at the same reaction conditions as above in 1 mL scale. The activity was calculated based on conversion of OA after 24 h reaction (TAN).
3.4. Concluding remarks It was shown in our earlier study that esterification of the triol, TMP with oleic acid catalysed by N435 had a lower environmental impact and also yielded a product with good biolubricant properties in comparison to that obtained with other heterogeneous catalysts [11]. This study analyses the effect of the process variables on the reaction efficiency and shows that high temperature and high biocatalyst loading as well as a stoichiometric excess of oleic acid are favourable for the reaction. Lipase catalysed synthesis of TMP-trioleate is expected to involve OA as the acyl donor (OA) and a number of acyl acceptors, i.e. TMP, TMP-monoester and TMPdiester, as the reaction progresses. These polyol esters may also function as acyl donors, and water acts as the acyl acceptor under the conditions of incomplete water removal that would result in hydrolysis of the esters. C. antarctica lipase B, the enzyme used in this study, has a large acyl donor site but a narrow acyl acceptor site [18], that may restrict the binding of the TMP-ester intermediates. The reaction was thus observed to take place with relatively high efficiency until the formation of diester product and is thereafter affected probably due to the bulkiness of the diester. The high temperature required for the reaction and also the polyol seem to have a detrimental effect on the enzyme activity, resulting in limited biocatalyst reusability and economic feasibility. Considering the low market price for speciality chemicals like biolubricants (in the range of 1 Euro/kg) and high cost of N435 (900 Euros/kg) at least 30-fold improvement in the biocatalyst activity/stability is estimated to be necessary to reach an economical process. Different strategies may be used to reduce the cost and improve the stability of the lipase. It has earlier been shown in our laboratory that the choice of a suitable matrix and optimising the amount of enzyme immobilised on the matrix can significantly influence the biocatalyst cost [19]. It was also suggested that increasing the scale of the process could drastically reduce the production cost of the biocatalyst [19]. Improvements can also be made by choosing or developing an enzyme with optimal molecular features such as higher stability to temperature and the polyol. For example, C. antarctica lipase B variant with enhanced thermostability at 90 ◦ C has been obtained
by random mutagenesis using an error prone PCR approach [20]. Another approach being investigated by us is to widen the acyl acceptor site so as to accommodate the TMP mono- and diesters and hence lead to a shorter reaction time and thereby less exposure of the enzyme to the harsh process conditions. Acknowledgments This work was performed within the framework of Greenchem – Speciality Chemicals from renewable resources, a research programme at Lund University, Sweden, supported by the Foundation for Strategic Environmental Research (Mistra). Roger Jacobsson, Jerker Johansson and Yvonne Samuelsson at AAK Sweden AB (Karlshamn, Sweden) are gratefully acknowledged for valuable discussions as well as for their help with analyses, product refining and evaluation. References [1] Willing A. Lubricants based on renewable resources – an environmentally compatible alternative to mineral oil products. Chemosphere 2001;43:89–98. [2] Schneider MP. Plant-oil-based lubricants and hydraulic fluids. J Sci Food Agric 2006;86:1769–80. [3] Ridderikhoff H, Oosterman J. Biodegradable hydraulic fluids: rheological behaviour at low temperatures of several oleochemically derived synthetic esters. Synth Lubr 2005;21:299–313. [4] Mang T, Dresel W. Lubricants and lubrication. Weinheim; 2001. [5] Fuchs Petrolub A-G. Synthetic lubricants and hydraulic fluids prepared by electrophilic addition of carboxylic acids to unsaturated fatty acids or esters. Germany DE Patent WO 2001053438; 2001. [6] Henkel K.-G.a.A., Transesterification method for the preparation of fatty acid polyol esters from triglycerides and polyhydric alcohols. Germany DE Patent WO 9957092; 1998. [7] Linko YY, Tervakangas T, Lamsa M, Linko P. Production of trimethylolpropane esters of rapeseed oil fatty acids by immobilized lipase. Biotechnol Tech 1997;11:889–92. [8] Monot F, Benoit Y, Vallerini D, Vandecasteele JP. Enzymatic synthesis of neopentylpolyol esters in organic media. Appl Biochem Biotechnol 1990;24–25:375–86. [9] Uosukainen E, Linko Y-Y, Lamsa M, Tervakangas T, Linko P. Transesterification of trimethylolpropane and rapeseed oil methyl ester to environmentally acceptable lubricants. J Am Oil Chem Soc 1998;75:1557–63. [10] People Republic of China. Process for synthesizing polyol ester with lipase catalyst. CN Patent CN 101475467; 2009.
C.O. Åkerman et al. / Process Biochemistry 46 (2011) 2225–2231 [11] Åkerman CO, Gaber Y, Abd Ghanid N, Lämsäe M, Hatti-Kaul R. Clean synthesis of biolubricants for low temperature applications using heterogeneous catalysts. J Mol Catal B Enzym 2011;72:263–9. [12] Chen B, Hu J, Xie W, Cai M, Gross R. Candida antarctica lipase B chemically immobilized on epoxy-activated Micro and nanobeads: catalysts for polyesters synthesis. Biomacromolecules 2008;9:463–71. [13] Goderis HL, Ampe G, Feyten MP, Fouwe BL, Guffens WM, Van Cauwenbergh SM, et al. Lipase-catalyzed ester exchange reactions in organic media with controlled humidity. Biotechnol Bioeng 1987;30:258–66. [14] Garcia T, Coteron A, Martinez M, Aracil J. Kinetic model for the esterification of oleic acid and cetyl alcohol using an immobilized lipase as catalyst. Chem Eng Sci 2000;55:1411–23. [15] Watanabe Y, Pinsirodom P, Nagao T, Yamauchi A, Kobayashi T, Nishida Y, et al. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase. J Mol Catal B Enzym 2007;44:99–105.
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[16] Hagström AEV, Nordblad M, Adlercreutz P. Biocatalytic polyester acrylationprocess optimization and enzyme stability. Biotechnol Bioeng 2009;102: 693–9. [17] Hilterhaus L, Thum O, Liese A. Reactor concept for lipase-catalyzed solvent-free conversion of highly viscous reactants forming two-phase systems. Org Process Res Dev 2008;12:618–25. [18] Naik S, Basu A, Saikia R, Madan B, Paul P, Chaterjee R, et al. Lipases for use in industrial biocatalysis: specificity of selected structural groups of lipases. J Mol Catal B Enzym 2010;65:18–23. [19] Tufvesson P, Törnvall U, Carvalho J, Karlsson A, Hatti-Kaul R. Towards a costeffective immobilized lipase for the synthesis of specialty chemicals. J Mol Catal B Enzym 2011;68:200–5. [20] Chodorge M, Fourage L, Ullmann C, Duvivier V, Masson JM, Lefevre F. Rational strategies for directed evolution of biocatalysts: application to Candida antarctica lipase B (CALB). Adv Synth Catal 2005;347:1022–6.