Combi-protein coated microcrystals of lipases for production of biodiesel from oil from spent coffee grounds
© Banerjee et al.; licensee Chemistry Central Ltd. 2013
Received: 12 May 2013
Accepted: 5 August 2013
Published: 8 August 2013
Replacing chemical catalysts with biocatalysts is a widely recognized goal of white biotechnology. For biocatalytic processes requiring low water containing media, enzymes for example commercial preparations of lipases, show low catalytic efficiencies. Some high activity preparations for addressing this concern have been described. Protein coated microcrystals (PCMC) constitute one such preparation. The present work describes a Combi-PCMC for synthesis of biodiesel from the oil extracted from spent coffee grounds.
Different lipases were screened for biodiesel synthesis from crude coffee oil out of which Novozym 435 gave the best conversion of 60% in 4 h. Optimization of reaction conditions i.e. % water, temperature and purification of coffee oil further enhanced conversion upto 88% in 24 h. A mixture of Novozym 435 and a cheap commercially available 1,3-specific lipase RMIM (from Mucor miehei) was used in different ratios and 1:1 was found to be the best trade-off between conversion and cost. The commercial preparations then were replaced by a novel biocatalyst design called Combi-Protein coated microcrystals (Combi-PCMC) wherein CAL B and Palatase were co-immobilized with K2SO4 as the core and this performed equivalent to the commercial preparations giving 83% conversion in 48 h.
Coffee oil extracted from spent coffee grounds could be used for the synthesis of biodiesel by using appropriate commercial preparations of lipases. The expensive commercially immobilized preparations can also be replaced by a simpler and inexpensive immobilization design called combi-PCMC which synergizes the catalytic action of a nonspecific lipase CAL B and a free form of 1,3-specific lipase from Mucor miehei.
KeywordsBiodiesel Protein coated microcrystals Coffee oil Lipases Transesterification
Lipases constitute the most frequently used enzyme in industrial enzymology [1–3]. Many industrial preparations are available commercially from several vendors. This ready availability of such preparations is largely due to early industrial application of lipases in fat splitting . In recent years, lipases have also been used in low water containing organic media for esterification/ transesterification reactions [5, 6]. In general “straight from the vendor” and lyophilized powders show poor catalytic activity in such media. This is due to the structural changes in the enzyme molecules which take place during the drying process [7, 8]. However, crystallization and precipitation have been found out to be better option for “drying” enzymes for use in low water media [9–16]. One such approach is preparation of protein coated microcrystals [10, 14]. In this biocatalyst design, enzyme gets precipitated over microcrystals of salts or amino acids or sugar molecules. Fairly high initial rates for catalysis in low water containing organic media have been obtained for PCMCs of horse radish peroxidase (HRP), soybean peroxidase (SBP), horse liver alcohol dehydrogenase (HLADH), catalase and lipases . For example, PCMC of lipase from Candida antarctica (CAL B) and Rhizomucor miehei (Palatase) showed a 5 and 15 fold increase in catalytic rates as compared to “straight from the vendor” preparations in the kinetic resolution of phenyl ethanol .
Major applications of lipases involve biotransformations involving fats and oils. In the context of such reactions, lipase can be classified either as 1, 3 specific (preferentially attacking 1 and 3 position on the triglycerides) or nonspecific (showing no preference to either 1 or 2 or 3 positions on the triglycerides). The choice of lipases, for example, during production of biodiesel, is largely governed by cost of lipase and its above mentioned specificity.
The present work involves preparation of combi-PCMCs of two different lipases for preparation of biodiesel starting with the oil obtained from spent coffee grounds. The two component lipases were palatase (lipase from Rhizomucor miehei in free form) and CAL B (lipase from Candida antarctica, also in free form). CAL B has been found to be a non-specific lipase when used for biodiesel formation . On the other hand, Rhizomucor miehei lipase is known to be a 1,3-specific enzyme .
The global shortage of fossil fuels and environmental concerns has sustained the interest in biodiesel production [19–30]. Biodiesel is a mixture of esters of long chain fatty acids and short chain monohydric alcohols (MeOH or EtOH). Biodiesel can be obtained from plant oils which constitute renewable resources. Biodiesel is biodegradable and its use (in place of diesel) leads to lower engine exhaust emissions of particulate matter and green house gases such as CO, CO2 and SOx[24, 30].
After the initial enthusiasm, there have been some concerns about the overall sustainability (of biodiesel production) as well as the diversion of the land (which could have been used for growing food crops) to growing energy crops . With regard to the latter context; using feed stock which is a waste material seems to be the most attractive idea. Such an approach solves the waste disposal problem and simultaneously yields a valuable product. The use of spent restaurant grease/oils for producing biodiesel has been one such approach [32–34].
Few years back, oil obtained from spent coffee grounds (the material which is left after the brewed coffee liquor is used for drinking) has been shown to be a viable source for preparing biodiesel [35, 36]. Recently, Calixto et al. have used supercritical methanol to carry out in situ extraction and transesterification of spent coffee ground oil to obtain biodiesel .
It has been pointed out that spent coffee grounds contain 10–15 weight % oil . With the global coffee production quoted as 16.34 billion pounds per year , spent coffee grounds constitute a huge amount of waste material. With chains of cafés exclusively devoted to serving coffee, the working model could be similar to the one adopted for collecting restaurant grease for producing biodiesel [32–34].
Biodiesel is a mixture of monoalkyl esters of fatty acids and is obtained by the transesterification of oils/fats. The transesterification can be catalyzed by either an acid or alkali or by an enzyme. When an enzyme is used as a catalyst, the reaction can be carried out at a moderate temperature; downstream processing is easier and if the feedstock oil contains high FFA content, enzymatic process seems to be more robust to deal with it [19–25]. Lipases have been used for this purpose and the process has been described with a very large number of oils and fats with varying degree of success [39–46].
Transesterification reactions catalyzed by lipases are carried out in a low water media [47, 48]. Either one can use nearly anhydrous organic solvents as reaction medium or work with a solvent free media in which case substrates (oil and the alcohol) constitute the reaction medium [20, 42, 45].
The present work shows that enzymatic transesterification does work fairly well with the relatively new and promising feedstock of spent coffee grounds. The catalysis could be carried out by employing a mixture of two commercially available immobilized preparations of two lipases. It was also shown that a new biocatalyst design called Combi-PCMC (combined protein coated micro crystals) could replace the above enzyme preparations as a less expensive alternative.
Results and discussion
Extraction of coffee oil
It has been reported that on an average spent coffee grounds yield about 11-20% oil . For oil extraction, simple hexane extraction under reflux conditions gave only 6% coffee oil. However when the hexane extraction was done in the soxhlet apparatus, the yield of coffee oil obtained was 14%. Three phase partitioning  using tert-butanol was also performed, which resulted in a 4% coffee oil recovery. It was decided to use the soxhlet method for obtaining the coffee oil in the present work.
Screening of lipases for biodiesel preparation from crude coffee oil
Effect of water content on biodiesel production
Effect of temperature on biodiesel preparation
Effect of enzyme loading
The cost of the enzyme is an important consideration in the overall process economics. It was seen that with 5% (w/w) enzyme, conversion to 70% esters was observed in 16 h. continuing the reaction upto 24 h did not result in any further conversion showing that the reaction had reached the equilibrium by 16 h (Additional file 1: Figure S1). As compared with 88% conversion with 10% (w/w) enzyme in 8 h (Figure 3), this was less efficient. Hence, reducing the amount of enzyme was not desirable if one wanted to achieve the maximum possible ester content. Further optimization experiments were carried out by using 10% (w/w) enzyme only.
Lipase catalysed biodiesel formation is controlled by a fairly complex set of parameters. The substrates, alcohol and the product glycerol are known to inhibit lipases [53–55]. In low water media, even water molecules act as enzyme inhibitors . Apart from these complex inhibition patterns, lipases also undergo inactivation. Hence, while transesterification reactions are believed to be equilibrium controlled, in reality the final % conversion is dependent upon the amount of enzyme used.
Purification of the coffee oil
Coffee oil is known to contain various low molecular weight substances . This results in coffee oil (as extracted) becoming a dark brown colored liquid. The oil was decolorized by refluxing with 2 g activated charcoal for 1 h. A comparison between the colored (crude) and decolorized (clean) oil was carried out to see if the polyphenols etc. present in the coffee oil had any effect on the percentage conversion to the ethyl ester.
Enzyme screening with clean coffee oil
It was seen that on the whole, decolourization of coffee oil with activated charcoal did not have a significant effect on the enzyme activity. However, all further work was carried out with the clean coffee oil.
Transesterification reaction with varying ratios of Novozym 435: RMIM
Novozym 435 is a relatively more expensive form of the commercially available lipase preparation. It was observed during the enzyme screening that the next best enzyme for biodiesel formation was RMIM, which is an immobilized form of the enzyme Rhizomucor miehei. This is relatively less expensive. However, as it is a 1,3-specific enzyme, some amount of Novozym 435 would be required for obtaining conversions beyond 66%. The presence of Novozym 435 and RMIM together were expected to give high conversion rates. The triglyceride would be transesterified by the 1,3-specific lipase to give the 2-monoglyceride. The 2-monoglyceride can also be transesterified by the non-specific lipase (CAL B) to give the fatty acid ethyl ester and glycerol. Therefore different ratios of RMIM and Novozym 435 were tried.
Designing inexpensive immobilized lipases for biodiesel synthesis
Spent coffee grounds were obtained from the local outlet of a chain of coffee cafeterias Cafe Coffee Day. The vendor uses a mixture of Arabica (Coffea arabica) and Robusta (Coffea canephora) coffee seeds and this was used as a starting material for extracting oil. Lipases from Pseudomonas cepacia [PS, PS C (lipase immobilized on ceramic particle), PS D (lipase immobilized on diatomaceous earth)], Candida rugosa (lipase AY), Lipase from Mucor javanicus: Lipase M; Lipase from Aspergillus niger: Lipase AP12; Lipase from Penicillium camemberti: Lipase G were kind gifts from Amano Enzymes (Nagoya, Japan). Lipase B from Candida antarctica (CAL B), Rhizomucor miehei (Palatase), Novozym 435 (immobilized form of CAL B), Rhizomucor miehei lipase immobilized on macroporous anion exchange resin (Lipozyme RMIM) were kind gifts from Novozymes, (Denmark). Ethanol was purchased from Merck (Germany) and n-hexane was purchased from Sigma Aldrich (St. Louis, USA). All the alcohols and solvents were further dried by keeping overnight over 3 Å molecular sieves bought from Merck (Mumbai, India).
Soxhlet extraction of oil from spent coffee grounds
Spent coffee grounds (50 g) were weighed in filter paper and placed in a 500 mL soxhlet glass timble. The extraction was carried out using n-hexane as solvent (380 mL) at 70°C. After extraction, the solvent was evaporated on a rotatory evaporator to obtain the coffee oil.
Spent coffee grounds (50 g) were added to hexane (400 mL) taken in a 1 L round bottomed flask and it was refluxed for 4 h. The hexane extract was filtered to remove the spent coffee grounds. The coffee oil was obtained from the filtered hexane extract by evaporating off the hexane in a rotatory evaporator.
Three phase partitioning (TPP) for oil extraction
Spent coffee grounds dispersed in water was mixed with 50% ammonium sulphate (w/v). tert-Butanol to aqueous layer was in a 1:1 ratio. It was then incubated for 1 h at 25°C. After 1 h the mixture was centrifuged at 6000 x g for 15 minutes at 25°C. The upper tert-butanol layer was then collected and dried using sodium sulphate. After drying the tert-butanol layer was evaporated on the rotary evaporator to remove the tert-butanol and obtain the coffee oil.
Decolourization of coffee oil
The coffee oil obtained by soxhlet extraction was refluxed with 2 g of activated charcoal at 70°C for half an hour. The hexane layer was then filtered to remove the activated charcoal. The clear hexane layer obtained after filtration was then evaporated in a rotary evaporator to remove the hexane and decolorized coffee oil was obtained.
Preparation of protein coated microcrystals (PCMCs) of the enzyme
PCMCs of CAL B and Palatase were prepared by starting directly from the liquid commercial preparations available from Novozyme, Denmark. The preparation of PCMCs was carried out essentially as described earlier . The activities of CAL B and Palatase preparations used in the present work were 6 U/mL and 125 U/mL respectively. The activity unit corresponded to the assay based upon hydrolysis of p-nitrophenyl palmitate [69, 70]. The corresponding protein contents were found to be 8.1 mg/mL and 6.8 mg/mL respectively as established by the dye binding assay . 100 μL of the enzyme was added to 100 μL of 10 mM sodium phosphate buffer pH 7.0. This solution was then added to 600 μL of a saturated solution of potassium sulphate. The resulting solution was added dropwise to 6 mL of ice chilled tert-butanol at 4°C with constant shaking followed by incubation at 4°C with a constant shaking at 200 rpm for 30 min. After completion of 30 min, the solution was first washed twice with tert-butanol and then once with acetone.
Preparation of combi-protein coated microcrystals (Combi-PCMCs) of Palatase and CAL B
Palatase (50 μL) and CAL B (50 μL) were added to 100 μL of 10 mM sodium phosphate buffer pH 7.0. This solution was then added to 600 μL saturated solution of potassium sulphate. The resulting solution was added drop-wise to tert-butanol at 4°C with constant shaking. The resulting solution was the incubated at 4°C with constant shaking of 200 rpm for 30 min. After completion of 30 min the solution was first washed with tert-butanol twice and then once with acetone.
Preparation of crosslinked combi protein coated microcrystals (CL-Combi-PCMCs)
The Combi-PCMCs obtained above were dispersed in 1 mL of tert-butanol. The Combi-PCMCs were crosslinked by incubating with 50 mM glutaraldehyde at 4°C with shaking at 300 rpm for 4 h. Combi-PCMCs thus obtained were washed twice with tert-butanol and then once with acetone.
Synthesis of biodiesel
Coffee oil (0.5 g) was mixed with ethanol (126 μL) in 4:1 molar ratio of ethanol: oil. The enzyme preparation was added to the reactant mixture followed by incubation at 40°C with shaking at 200 rpm. Aliquots were taken at different time intervals and the percentage conversion to ethyl ester was determined by carrying out the GC analysis of the samples.
GC analysis for biodiesel
The alkyl esters were analyzed on Agilent Technologies 6890 N network GC systems, USA with a flame ionization detector. The standard reference method EN 14103 was used . The capillary column HP- 5 (5% diphenyl 95% dimethylpolysiloxane), 30 m X 0.32 mm X 0.25 μm (Agilent) was used. Nitrogen was used as the carrier gas. The column oven temperature was programmed in the range of 150°C to 250°C at 10°C min-1 with injector and detector temperatures at 240°C and 250°C, respectively.
The resulting fatty acid esters (biodiesel) from the reaction were weighed and mixed with the internal standard methyl heptadecanoate (10 mg/ml solution in hexane). The final concentration of sample in the mixture is 50 mg/ml. 1 μl of this mixture was injected in the GC. Peak areas of fatty acid esters and internal standard were obtained.
ΣA = total peak area C14:0 – C24:1
A’ = internal standard peak area (methyl heptadecanoate)
C’ = concentration of internal standard solution in mg/mL
V’ = volume of internal standard solution used in mL
m = mass of the sample in mg
To sum up, coffee oil extracted from spent coffee grounds could be used for the synthesis of biodiesel by using appropriate commercial preparations of lipases. The expensive commercially immobilized preparations can also be replaced by a simpler and inexpensive immobilization design called combi-PCMC which synergizes the catalytic action of a nonspecific lipase CAL B and a free form of 1,3-specific lipase from Rhizomucor miehei.
The preparation of biodiesel employed a waste material as an oil source. The coffee plant itself is a renewable resource. The biocatalyst design used a simple salt as the immobilization matrix. Hence, the process constitutes a sustainable as well as an inexpensive approach for biodiesel synthesis.
JM thanks the Council of Scientific and Industrial Research for the Senior Research Fellowship. Funds obtained from the Government of India's Department of Science and Technology (DST) [Grant No.: SR/SO/BB-68/2010] and Department of Biotechnology (DBT) [Grant No.: BT/PR14103/BRB/10/808/2010] are gratefully acknowledged.
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