A rapid enzyme-catalyzed pretreatment of the acidic oil of macauba (Acrocomia aculeata) for chemoenzymatic biodiesel production

Accepted Manuscript Title: A rapid enzyme-catalyzed pretreatment of the acidic oil of macauba (Acrocomia aculeata) for chemoenzymatic biodiesel production Author: Danielle Altomari Teixeira C´esar Rezende da Motta Claudia Maria Soares Ribeiro Aline Machado de Castro PII: DOI: Reference:

S1359-5113(16)31073-X http://dx.doi.org/doi:10.1016/j.procbio.2016.12.011 PRBI 10883

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

30-8-2016 8-12-2016 17-12-2016

Please cite this article as: Teixeira Danielle Altomari, Motta C´esar Rezende da, Ribeiro Claudia Maria Soares, Castro Aline Machado de.A rapid enzyme-catalyzed pretreatment of the acidic oil of macauba (Acrocomia aculeata) for chemoenzymatic biodiesel production.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.12.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A rapid enzyme-catalyzed pretreatment of the acidic oil of macauba (Acrocomia aculeata) for chemoenzymatic biodiesel production

Short title: Pretreatment of macauba (Acrocomia aculeata) acidic oil

Danielle Altomari Teixeira, César Rezende da Motta, Claudia Maria Soares Ribeiro, Aline Machado de Castro*

Biotechnology Division, Research and Development Center, PETROBRAS, Av. Horácio Macedo, 950. Ilha do Fundão, Rio de Janeiro 21941-915, Brazil. Phone: +55 21 21622811. *

Corresponding author: E-mail: [email protected]

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Graphical abstract

Highlights: Acrocomia aculeata is a promising feedstock for biodiesel production. Lipozyme 435 (5 % w/w) reduced 91.5 % of macauba oil acidity in 4 hours. Enzyme was robust at a broad temperature range (20-45°C). Concomitant esterification and transesterification was observed.

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Abstract: Macauba (Acrocomia aculeata) is a plant with high potential of oil supply for biodiesel production. However, the high acidity (35–43%) of this oil disqualifies it from use in industrial biodiesel production plants that use alkaline transesterification as the synthesis reaction. Thus, technologies such as the use of biocatalysts to reduce oil acidity are necessary to better monetize the carbon present in that biomass. In the present study, a commercial lipase (Lipozyme 435) was used for the esterification of free fatty acids (FFAs) in the oil to reduce the acidity. Lipozyme 435 performed well under broad temperature (20–45°C) and water content (560–30000 ppm) ranges, and its performance was maximized in a substrate (methanol:FFA) molar ratio of 2. The lowest final FFA content achieved was 1.09%, corresponding to a global conversion rate of 97.22%. The fatty acid methyl esters content in the treated oil (55%), determined by 13C NMR, indicated that Lipozyme 435 can also catalyze the transesterification of the glycerides in the oil. The versatility under different operational conditions and stability over long use (up to 150 times) indicated that Lipozyme 435 is a very suitable biocatalyst for the proposed process, contributing to a better economic attractive option.

Keywords: Biodiesel; Lipase; Acrocomia aculeata; Enzymatic esterification; Acidic oil; Free fatty acid

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1. Introduction At present, the monthly total production of the 52 biodiesel plants in Brazil is approximately 320,000 m3, and from this total, 78% of the production comes from soybean oil. However, the cost of this raw material represents 83–91% of the entire biodiesel production cost, thus reducing the profit margin of the mill owners [1]. Therefore, it is necessary to use alternative raw materials with lower cost that do not compete with food production and present high availability for the production of biofuel [2]. For this purpose, the Brazilian Ministry of Agricultural Development, through the Biofuels General Coordination, has been seeking to diversify the oil raw materials used in the production of biofuels. Acrocomia spp. were identified as promising raw materials for biodiesel plants [3]. Among the Acrocomia spp., macauba (Acrocomia aculeata) shows high oil productivity and is well adapted to the broad Brazilian climate, from North to South. This plant can also be used in the recovery of degraded lands and agroecology intercropping, in addition to being carbon retentive and not being a traditional oil source consumed by humans [1–5]. In addition to oil production, wastes generated during macauba fruit processing can be used for the production of coproducts such as biogas, bio-oil, bio-kerosene, second generation ethanol, and solid biofuels to generate electricity and steam. Thus, this diversification in the production of other energy products adds value to macauba and contributes to the sustainability of the process [5,6]. It is estimated that the oil yield of commercial macauba crops is similar to that of the palm fruit (palm oil), which is approximately 4–6 ton oil/ha [7]. In Brazil, macauba oil is mainly produced in the state of Minas Gerais. However, its high acidity hinders its refining in biodiesel plants [8]. The high acidity of macauba oil is related to the pre- and postharvest conditions. The macauba production system is based on 4

extractivism, and fruits are collected from the ground. Then, the degradation is accelerated by soil microorganisms. After the harvest, the fruits are usually stored in uncontrolled environments and can be affected by exposure to light and humidity [6,7]. Oils with a high free fatty acid (FFA) content cannot be used in the process employed in most mills (homogeneous alkaline transesterification) to obtain biodiesel because of the formation of soap, yield loss, and increased difficulty in product separation [2,8,9]. In biodiesel mills that receive oils with less (<10%) FFA content, the acids are commonly removed from the oil through refining processes (physical or chemical) and then sold for a much lower value than that they would obtain if converted into fatty acid methyl esters (FAME) [10]. A traditional approach to produce biodiesel from acid feedstocks is to employ acid catalysis because such catalysts (e.g., sulfuric acid) are less sensitive to high FFA content; however, excess methanol (molar ratio of 8–200) is required [9,11, 12]. In addition to the environmental concerns, corrosion problems are also related to this route because acid (catalyst) content may vary from 4% to 90% of the FFA weight [9,11]. Enzymatic biocatalysts can be used as an environmentally friendly and efficient alternative treatment. Enzyme catalysts (biocatalysts) such as lipases (triacylglycerol acylhydrolase, EC 3.1.1.3.) have the potential to reduce oil acidity by converting FFA to FAME [10, 13]. The adoption of enzymes for oil pretreatment instead of the synthesis of biodiesel per se is because

enzyme-catalyzed

esterification

reactions

generally

proceed

faster

than

transesterification reactions [14-18], and in many cases, obtaining a fully specified biodiesel stream remains challenging for biocatalysis [19-20]. Therefore, the aim of this study was to investigate a lipase-catalyzed process for macauba oil acidity reduction with an aim to use the deacidified oil in industrial biodiesel plants equipped with facilities for alkaline transesterification.

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2. Materials and Methods 2.1. Materials The two macauba oil samples used in this study were obtained from COOPERRIACHÃO cooperative (Minas Gerais state, Brazil). The first one (named Oil 1) had an initial acidity of 42.6% and water content of 5000 ppm, and the second one (named Oil 2) had an initial acidity of 36.9% and water content of 3484 ppm. The immobilized enzyme Lipozyme© 435 was used as a biocatalyst for the reactions. It was purchased from LNF Latinoamericana, a representative of Novozymes in Brazil.

2.2 Reactions for acidity reduction Tests were performed to evaluate the oil acidity reduction through the conversion of FFA into FAME by enzymatic action. The reaction systems used consisted of jacketed batch reactors that were interconnected by hoses. The reactors were placed on a stir plate with magnetic stirring in a 50-mL scale reactor to promote a homogenization system. The water bath was connected to a water system and provided preset temperature to all the reactors. The amount of oil in each reactor ranged from 11–40 g, depending on the experiment. Thereafter, different operational conditions such as enzyme loading (0.1–25% w/w), temperature (20– 45°C), methanol:FFA molar ratio (1–3), and water content (560–30000ppm) were evaluated. The amount of oil employed was 20g for the evaluation of the effects of enzyme loading, temperature and molar ratio and 40g for the effect of water content. To investigate the effect of the water content on the reaction progress, the oil was previously dryed in a rotary evaporator until a water content of 268 ppm was obtained. It should be infomed that we just added external water in the experiment specific for evaluation of the effect of water content in the reaction. In all other cases, the water contained in the reactions was that original from the substrates (as informed in Section 2.1). 6

The pretreated oil was also used in a second reaction stage for improved acidity reduction where the effect of methanol addition was investigated. These reactions were performed at 30°C with 12 g of pretreated oil from the repeatability test (please see section 3.5) and specific conditions, as described below: 1. Condition “methanol (R=0)”: Addition of 5% (w/w) fresh enzyme and no addition of methanol; 2. Condition “methanol (R=1)”: Addition of 5% (w/w) fresh enzyme and addition of methanol to obtain a molar ratio of 1 (based on initial FFA content in the untreated oil); Reaction progress was monitored by aliquot withdrawal (between 400 and 800 μL) at different sampling times (0, 0.25, 0.5, 1, 2, 4, 6, and 24 h). The samples were centrifuged at 8517 ×g for 5 min. After centrifugation, the samples were weighed (approximately 0.08 g) and then dissolved in 55 ml of hydrous ethanol and titrated. All experiments were performed in duplicate, and analyses were performed at least in duplicate.

2.3 Biocatalyst stability Stability experiments were conducted to investigate the performance of Lipozyme 435 during long reaction times. The methodology adopted has been used for several years for catalyst evaluation in our company, and it multiplies the proportion between the ratio of the substrate mixture (oil and methanol, with a methanol:FFA ratio of 2) to the immobilized enzyme by n factors, thus extending the reaction time at the same proportion. In the present study, four factors were investigated (n=20, 50, 100, or 150) and applied over a 4-h reaction time of the repeatability condition (see section 3.5), resulting in total reaction times of 80, 200, 400, and 600 h, respectively. In each test, all the reagents (methanol, oil, etc.) and the

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enzyme were added at the beginning and were kept in contact during the extended reaction. No biocatalyst manipulation or transferring to another vessel occurred during the experiments, which was different from conventional enzyme reuse tests. The amount of oil used in each condition was 40g.

2.4 Analyses and calculations Macauba oil acidity (FFA content) was determined by neutralization titration in an automatic titrator (Metrohm 862, Herisau, Switzerland) contaning electrode for nonaqueous solutions using 0.04 mol/L NaOH until pH 11 (end point). The water content in the macauba oil was determined using a Karl Fischer (Metrohm 852 Titrando) titrator. Th econversion rate was calculated using the difference between the initial acidity (mean of the replicates) and the acidity at each time (x = value of each replicate) in the reaction, as shown in Equation 1.

𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛(%) =

(𝐴𝑐𝑖𝑑𝑖𝑡𝑦𝑖𝑛𝑖𝑡𝑖𝑎𝑙 −𝐴𝑐𝑖𝑑𝑖𝑡𝑦𝑡=𝑥 ) 𝐴𝑐𝑖𝑑𝑖𝑡𝑦𝑖𝑛𝑖𝑡𝑖𝑎𝑙

× 100

(Equation

1)

The composition of pretreated macauba oil was determined by 13C nuclear magnetic resonance (NMR). Samples (approximately 1g) were solubilized in CDCl3 and analyzed in an Agilent MR-400 equipment in 10-mm tubes. Spectra were recorded on Agilent 400MR (9.40 Tesla) NMR spectrometer. A radiofrequency pulse angle of 90 and a delay time of 1.3 s between pulses were applied during data acquisition. The

13

C NMR spectra were processed

by multiplying the free induction decay with an exponential function using a line-broadening factor of 1.0 Hz.

8

3. Results and discussion 3.1 Effect of enzyme loading In the initial studies, the performance of several commercial biocatalysts was evaluated (data not shown) and Lipozyme 435 was selected as the most promising enzyme. This biocatalyst was then used in different amounts (% w/woil) (Figure 1), and a satisfactory performance was observed using a 5% loading volume. In this condition, the acidity was reduced by 91.5% after 4 h of reaction. The use of 25% of biocatalyst resulted in an acidity reduction of 89% in 30 min of reaction. In a similar study, Souza et al. [21] reported higher FFA conversion (83.5%) with a longer reaction time (90 min) using soybean oil and Novozym 435 (3% w/w), with the addition of ethanol in two stages and at 50°C. Nordblad et al. [12] reported the enzymatic conversion of FFA to FAME before introducing it into the alkaline process to produce biodiesel, using rapeseed oil with up to 15% FFA content, Novozym 435 (5% w/w), and a molar ratio of 5.2 (methanol/FFA). However,

the acidity reduction achieved was

approximately 0.5%, which represents a much lower reduction than that in the present study. In the present study, we used a feedstock with higher FFA content (35–43%) and obtained significant results with a lower methanol excess (molar ratio of 2 for methanol/FFA) and a lower temperature (30°C), thus contributing to the environmental and economic advantages in the said process.

3.2 Effect of temperature At a biocatalyst loading volume of 5% (w/w), its performance was evaluated at different reaction temperatures that included typical climatic conditions of the regions where macauba is cultivated in Brazil. The results presented in Figure 2 indicate that the biocatalyst showed robust performance in the evaluated range, suggesting that there is no need for an 9

heat exchange system for maintaining the reaction temperature; this finding further favors the economics of the process described here. Lipozyme 435 can also act in the presence of higher temperatures (60–110°C) that are used for the esterification reaction of linoleic acid with oligoglycerol (molar ratio of 1:1.5) [22]. The robust performance of Lipozyme 435 was also reported by Souza et al. [21] and Ortega et al. [23], and its use as a biocatalyst in esterification reactions was also reported by Duan et al. [24].

3.3 Effect of the methanol/FFA molar ratio The effect of the methanol/FFA molar ratio was investigated in the range of 1-2.5, including stepwise alcohol addition (overall molar ratio of 3, with 2/3 added at the beginning of the reaction and 1/3 added after 30 min of reaction) (Figure 3). The methanol stepwise addition strategy was performed with an aim to provide an excess of alcohol to shift the reaction towards FAME synthesis, while simultaneously avoiding inhibitory levels to the enzyme. The lowest acidity after 6 h of reaction (2.9%) was obtained when the stepwise addition was adopted and when the methanol/FFA molar ratio of 2.5 was used. In this latter case, however, there was a slight decrease in the reaction rate at the beginning of the reaction, which may be attributed to enzyme inhibition. Acid conversion when the methanol/FFA molar ratio of 2 was used (90.7±0.8 %) was very close to the conversion of the stepwise addition (92.4±0.3%); therefore, it was decided to maintain the molar ratio of 2 for subsequent tests. When equimolar quantities of methanol and FFA were employed, the reaction occured during 1 h and later stopped, indicating that an excess of methanol is necessary to proceed the reaction toward higher conversion. Zhou et al. [25] reported a stepwise methanol addition for the deacidification of soybean oil deodorizer distillate at 29°C, catalyzed by Candida rugosa lipase. Methanol (molar ratio of 1) was added initially, and then after 3, 6 and 9 h resulting in a total molar

10

ratio of 4. The highest conversion (90.2%) was achieved after 18 h of process. C. rugosa lipase was also employed for an acid oil FFA conversion in the study of Watanabe et al. [26]. After 24h of reaction at 20°C with a methanol/FFA molar ratio of 5 and 1% w/w lipase, the degree of esterification was 96%.

3.4 Effect of initial water content The robust performance of Lipozyme 435 was also demonstrated over a broad range of initial water content (Figure 4), a typical variability that can be found in oils from different cooperatives. A slight difference was observed in enzyme performance in the presence of macauba oil with varied water content in the reaction. However, it was expected that high water content could favor hydrolysis reaction, resulting in more FFA, and thus decreasing the efficiency of the enzymatic reaction [27-30]. Prevous studies also observed a robust enzyme performance during reactions with water content of up to 10% (v/v) [31], which was suggested to be related to the formation of an interface more favorable for the active conformation of the enzyme.

3.5 Evaluation of test repeatability The evaluation of the repeatability of oil acidity reduction reaction in the condition that showed the best results indicated a small variation between the replicates. The reaction was carried out using Lipozyme 435 (5% w/w), molar ratio methanol/FFA of 2, and at 30°C. The overall result of the eight tests (duplicate analysis of the four experimental replicates) indicated final average acidity of 3.24±0.29% and 91.7±0.7% conversion.

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The composition of pretreated macauba oil from ‘Oil 1’ batch was determined by 13C NMR. The contents of FAME, methanol, triglycerides, diglycerides and total glycerine were estimated to be 55.0%, 1.0%, 40.0%, 4.1%, and 4.9%, respectively. Monoglycerides were under detection limit. It was observed that the final FAME content was higher than initial FFA content in the reaction (39.29±3.24%), which indicates that Lipozyme 435 could catalyze alcoholysis reaction (transesterification) of glyceride oil and not just esterification of FFA.

3.6 Second reaction stage To achieve the lowest possible final oil acidity, two strategies were evaluated in the second reaction stage, including addition of fresh enzyme and addition of further quantity of methanol (Figure 5). The best result was observed when the additional methanol/FFA molar ratio of 1 was used (condition ‘Methanol R=1’) resulting in a minimal acidity of 1.09±0.31% and a maximal conversion (considering the first and the second stages) of 97.22±0.79, after 60 min reaction. In this condition, however, reaction reversibility was subsequently detected, possibly due to the water present in the reaction (as an esterification product). When only fresh enzyme was added, without methanol addition (condition ‘Methanol R=0’), a less extent of acidity reduction was observed until 45 min of reaction, followed by hydrolysis. Thus, from the results obtained in this test, it can be concluded that it is important to ensure that a sufficient amount of methanol is available to shift reaction toward FAME synthesis. This is in accordance with the clear effect of the methanol/FFA molar ratio on the reaction progress, as previously observed in this study (Figure 3).

3.7 Biocatalyst stability

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Finally, biocatalyst stability was evaluated, and the results (Table 1) suggest that the enzyme may have potential to be reused for a medium-to-long time for this reaction. This is a preliminary evaluation of operational stability, since the enzyme is exposed to different conditions that were used in the reaction (longer time in contact with higher methanol concentration, since enzyme loading is significantly lower and the reaction takes longer to proceed), but it is a very practical initial evaluation that avoids interferences such as weight loss of the biocatalyst due to enzyme manipulation between cycles. Moreover, this approach does not use costly solvents for biocatalyst washing, which is commonly adopted in enzyme reuse studies. After 20, 50,100 and 150-times longer reaction, the final oil acidity was 5.28±0.35%, 6.54±0.48%, 6.15±0.24% and 6.53±0.23%, respectively, which are already suitable values to specify the pretreated oil as charge for a typical physical refining used in industrial biodiesel plants.

Similar to the present study, Chen et al. [32] used an immobilized lipase (Novozym 435) over 30 days without loss of catalytic activity in a packed-bed reactor for the continuous production of biodiesel by methanolysis of soybean oil in the presence of tert-butanol. A high molar conversion (80%) was obtained with a flow rate of 0.1 mL/min at 52.1◦C and methanol molar ratio of 4. In a different approach, Aguieiras et al. [33] reused three different commercial immobilized lipases (Novozym 435, Lipozyme RM IM, and Lipozyme TL IM) in ethanolysis of soybean oil for biodiesel production; in this study, the effect of differents solvents were evaluated for use in the washing of immobilized lipases before the reuse of the enzyme. Butanol and ethanol led to the lowest reduction in ester yield after the first cycle, which was associated to better glycerol removal from the biocatalyst surface. On the other hand, hexane was shown by Scanning Electron Microscopy analyses to affect the support structure of the biocatalyst. Novozym 435 reuse was also investigated by Kochepka et al.

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[34]. The biocatalyst was washed with either a tert-butanol/water mixture or with only tertbutanol, after 10 h of reaction for fatty acid ethyl esters (FAEE) production from waste cooking oil, at 40°C, using 5% w/w of biocatalyst and ethanol/oil molar ratio of 9. FAEE content at the end of the reaction decreased from 76.5% in the first cycle to 31.9% (when the solvents mixture was used) and 73.5% when only the organic solvent was employed. Stability was not studied for more enzyme reuses. Thus, whenever possible, it seems an interesting approach (from both technical and economic points of view) to avoid the use of solvents for enzyme reuse.

Conclusions Heterogeneous biocatalysis presents potential for oil acidity reduction. In this sense, high-acidity oils can be used in existing biodiesel mills containing facilities for the conventional alkaline transesterification process, after a rapid enzyme-catalyzed pretreatment that converts FFA into FAME. In the present study, we demonstrated that Lipozyme 435 show robust catalytic performance over broad temperature and initial water content ranges, thus representing economical advantages for the implementation of this route. The lowest final oil acidity achieved (1.09%) with the enzymatic treatment implies that the pretreated oil would require only a simple (established) chemical refining before entering the alkaline transesterification reactor. Enzyme stability was considered satisfactory, and additional enzyme reusability tests are suggested to determine enzyme efficiency when exposed to the best reaction conditions described here for a long time.

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Acknowledgments The authors would like to acknowledge: PETROBRAS for the financial support; Sonia Cabral for her support in NMR analysis; and Márcio Portilho, Alexander Bastos, and Vitor Ximenes for their support in Karl Fischer analysis.

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Figures captions Figure 1: Effect of different Lipozyme 435 concentrations on the acidity reduction (A) and FFA conversion (B) of macauba oil. Reaction conditions: Oil 1 (20g); initial methanol/FFA molar ratio, 2; and temperature, 30ºC. Figure 2: Effect of temperature on the acidity reduction of macauba oil. Reaction conditions: Oil 1 (20g); initial methanol/FFA molar ratio, 2; and Lipozyme 435 content, 5% w/w. Figure 3: Evaluation of different initial methanol/FFA molar ratio on the acidity reduction (A) and FFA conversion (B) of macauba oil. Reaction conditions: Oil 1 (20g); temperature, 30°C; and Lipozyme 435 content, 5% w/w. Condition ‘3(2/1)’ indicates total methanol/FFA molar ratio of 3, with 2/3 at the beginning and 1/3 after 30 min of reaction. In all other conditions, methanol was completely added at the beginning of reaction. Figure 4: Evaluation of different water concentrations for acidity reduction of macauba oil Reaction conditions: Oil 2 (40g); temperature, 30°C; initial methanol/FFA molar ratio, 2; and Lipozyme 435 content, 5% w/w. Figure 5: Evaluation of the second reaction stage for acidity reduction (A) and FFA conversion (B) of macauba oil. The first reaction stage was carried out according to repeatability test (Oil 1; temperature, 30°C; initial methanol/FFA molar ratio, 2; and Lipozyme 435 content, 5% w/w), and temperature was kept the same for the second stage. Conditions investigated in the second stage: mass of oil: 12g; in ‘methanol (R=0)’, only 5% w/w of fresh enzyme was added; in ‘methanol (R=1)’, 5% w/w of fresh enzyme and methanol for a molar ratio of 1 (based on initial FFA content in the untreated oil) were added.

20

Figure 1

A)

100

Conversion (%)

80 60 40 20 0

B)

0

4

8 12 16 Reaction Time (h)

20

24

21

Figure 2

50

20°C 35°C 45°C

Acidity (%)

40

30°C 40°C

30 20 10 0 0

1

2 3 4 Reaction Time (h)

5

6

22

Figure 3

50

R=1 R=2 R= 3(2/1)

Acidity (%)

40

R=1.5 R=2.5

30 20 10 0 0

A)

1

2

3 4 Reaction Time (h)

5

6

2 3 4 Reaction Time (h)

5

6

100

Conversion

80 60 40 20 0

B)

0

1

23

Figure 4

50

560 ppm 1000 ppm 3000 ppm 5000 ppm 10000 ppm 15000 ppm 20000 ppm 30000 ppm

Acidity (%)

40 30 20 10 0 0

1

2

3 4 Reaction Time (h)

5

6

24

Figure 5

A)

B)

25

Table 1: Lipozyme 435 (5% w/w) stability during long-time reaction for acidity reduction of macauba oil (Oil 2, 40g) at 30ºC and a methanol/FFA molar ratio of 2. Factor n

1

Total reaction time

Final conversion (%)

Reaction eficiency (%)1

20

80h

86.5±0.9

94.3

50

200h

83.4±1.2

90.9

100

400h

84.6±0.6

92.2

150

600h

82.7±0.6

90.2

Reaction eficiency was compared to that observed after 4h in the reaction at optimized conditions

(methanol/FFA molar ratio of 2, 30°C, 5% (m/m) Lipozyme).

26