Biocatalysis: An Industrial Perspective

Research and Development Center, PETROBRAS, Av. Horácio Macedo, 950, Ilha do Fundão, Rio de Janeiro 21941-915,Brazil

10.2 Hydrolysis of Lignocellulosic and Starchy Biomass

Sugarcane bagasse is one of the main agroindustry lignocellulosic biomasses in the world. In Brazil alone, its generation at mills has been over 100 million tons since 2000.³ This biomass can be used for either the production of tailor-made enzyme cocktails or as substrate for enzymatic hydrolysis to generate fermentable sugars. Agroindustrial lignocellulosic biomass is mainly constituted of three fractions: cellulose (15–55 wt%), hemicellulose (25–50 wt%) and lignin (10–40 wt%).⁴ In most cases, hemicellulose is removed from the solid fraction and often depolymerized during physico-chemical pretreatments,⁵ resulting in a solid that consists mostly of cellulose and lignin, which is named cellulignin by some authors.^6–8 The deconstruction of cellulose present in cellulignin involves primarily the action of three groups of enzymes: cellobiohydrolases (EC 3.2.1.91), endoglucanases (EC 3.2.1.4) and β-glucosidases (EC 3.2.1.21) (Figure 10.1(a)), with specificities for different parts of the polysaccharide.³ These enzymes act synergistically for the release of the final fermentable sugar, glucose. Figure 10.1(b) represents a type of synergy between cellobiohydrolases and β-glucosidases with sequential release of cellobiose (glucose β-1,4-linked disaccharide) and then glucose, whereas Figure 10.1(c) shows synergy between endoglucanases and β-glucosidases, indicating that the last enzymes can also catalyze hydrolysis of oligossacharides.^3,9

Figure 10.1 Representation of cellulases synergistic action. (a) Global synergy; (b) CBH/BG synergy, and (c) EG/BG synergy. EG: endoglucanase; BG: β-glucosidase; CBHI: cellobiohydrolase I; CBHII: cellobiohydrolase II. Black and white circles correspond to reducing and non-reducing glucose units, respectively. Figure adapted with permission from ref. 3.

Cenpes’ research for deconstruction of sugarcane bagasse started in 2004, in partnership with the Federal University of Rio de Janeiro. This biomass was used for the production of cellulases by several fungi,¹⁰ with promising results with Penicillium funiculosum,^11,12Trichoderma harzianum^13,14 and Aspergillus niger,¹³ each one with a distinct profile of cellulolytic activities (Figure 10.2).

Figure 10.2 Activity of cellulolytic enzymes produced by filamentous fungi. Bubble size corresponds to cellobiohydrolase activity (numerical activity is given after the fungus name).

As a part of cooperation with PETROBRAS, a preliminary study investigated cellulase production by P. funiculosum using either untreated or pretreated (only acid, only alkali and acid plus alkali pretreatments) sugarcane bagasse as carbon source and inducer.¹¹ Acid pretreatment increased substantially carboxymethyl cellulose (CMCase, endoglucanase) activity, whereas filter paper (FPase) and β-glucosidase activities were mainly improved by alkali pretreatment. The highest FPase, β-glucosidase and CMCase activities were 354, 1835 and 3588 U L⁻¹, respectively. Later, Maeda et al.¹⁵ optimized the culture conditions of this same strain and the CMCase activity increased to 6917 U L⁻¹, with significant improvements in productivity, achieved within 72 h of fermetnation.

More recently, studies on lignocellulose deconstruction carried out by PETROBRAS included accessory proteins, such as swollenins and lytic polysaccharide monooxygenases (LPMOs),^9,16 as well as cellulosomes^9,17 and addition of other non-hydrolytic proteins, such as bovine serum albumin combined with surfactants.¹⁸ Rocha¹⁶ expressed a T. harzianum swollenin in A. niger, achieving a protein concentration of 197 mg L⁻¹, which is around 93 times higher than that found in the parent strain. The use of this T. harzianum protein increased by two-fold the hydrolysis efficiency of a commercial cellulase cocktail, on bench-scale preliminary tests.¹⁹

The enzymes produced by selected filamentous fungi using sugarcane bagasse as inducer were applied in cellulose hydrolysis. The cocktail from P. funiculosum boosted the activity of the commercial cellulase preparation Multifect©, and the combined enzymatic hydrolysis was increased by 50%.²⁰ In another set of experiments, an experimental mixture design was applied for optimization of acid and alkali pretreated sugarcane bagasse hydrolysis. By using a proportion of 15% of T. harzianum IOC-3844, 50% of P. funiculosum ATCC11797 and 35% of A. niger ATCC 1004 cocktails, a global hydrolysis yield of 91% was achieved after 48 h.²¹

The one-pot conversion of sugarcane bagasse into ethanol in a consolidated bioprocessing strategy was preliminarily studied by Groposo et al.¹⁷ The strain Clostridium thermocellum ATCC27405 was able to produce a slightly higher concentration of the alcohol when cultivated in the presence of raw sugarcane bagasse (21.9 mM) than that observed when the corresponding acid pretreated biomass was used (19.6 mM), after 72 h of fermentation. This proof of concept opens new perspectives, since it indicates that biomass pretreatment is not always necessary for second-generation ethanol production.

Finally, PETROBRAS is also involved in the conversion of the sugars from enzymatic hydrolysis for the production of molecules of interest by industry, via fermentation. Initially, the interest was focused on ethanol. Many publications were reported as a result of this interest, including pretreatment strategies^22–24 and fermentation.²⁵ More recently, other bioproducts were also targeted, such as succinic acid,²⁶ butanol,²⁷ and propionic acid.²⁸ At Cenpes facilities, biomass deconstruction studies have been carried out since 2004 at bench scale and since 2007 at a small pilot scale. PETROBRAS has a patent portfolio related to lignocellulose deconstruction. The granted patents are summarized in Table 10.1. In addition, there are other three patent applications under consideration (PI 0803782-5, PCT/BR2010/000216 and PCT/BR2010/000334).

Table 10.1 Granted patents related to lignocellulose deconstruction

Company	Company
Process for producing ethanol from a hydrolysate of the hemicellulose fraction of sugarcane bagasse in a press reactor	PI0505299-8 (Brazil), US8642289 B2 (USA), EP2167672 B1 (EPO), ES2382001 (Spain), DK2167672 (Denmark)
Process for the fermentative production of ethanol from solid lignocellulosic material comprising a step of treating a solid lignocellulosic material with alkaline solution in order to remove the lignin	DK178525 (Denmark), JP5325793 (Japan), SE535902 C2 (Sweden), CA2660673 (Canada), GB2454119 (Great Britain), US8232082 B2 (USA)
Process for production of an enzymatic preparation for hydrolysis of cellulose from lignocellulosic residues and application thereof in the production of ethanol	EP2373787 B1 (EPO)

For the production of biomolecules from lignocellulosic biomass, there are still some challenges that need to be addressed. From the technical point of view, improvements in pretreatment procedures for a reliable industrial operation must be considered. An efficient pretreatment also tends to influence both hydrolysis (by means of cellulose accessibility to enzymes) and fermentation efficiencies (regarding an inhibitor’s – acetic acid, furfural, hydroxymethylfurfural – concentration). The exploration of the synergy between cellulolytic enzymes, oxidative enzymes and accessory proteins, and the production of a cocktail at low cost, is equally important and may drastically impact process economics. Finally, the use of microbial strains (either recombinant or not) with the ability to produce efficiently the bioproducts from both C₅ (xylose, arabinose) and C₆ (glucose) sugars, under a stable metabolic activity, is of paramount importance to achieve feasible technical and economic targets. PETROBRAS has been also working on this topic, in collaboration with the University of Brasilia.29

It is worth mentioning that PETROBRAS also had a strong development regarding ethanol production from starchy biomass. This interest actually started during the 1970s and then returned early this century with two approaches. One was the application of commercial enzymes for sequential liquefaction and saccharification of castor bean cake (Ricinus communis L.) followed by alcoholic fermentation, in which a hydrolysis efficiency of 91.4% and ethanol titer of 34.5 g L⁻¹ were obtained.^30–32 The other was the production of combined tailor-made amylolytic/proteolytic cocktails from agricultural cakes and brans^33–39 and their use for cold starch hydrolysis (hydrolysis at sub-gelatinization temperature) and ethanol production, in which an ethanol titer of 78 g L⁻¹ was obtained.^40–42

10.3 Synthesis of Solvents

PETROBRAS research and development in biocatalysis for the synthesis of solvents focuses on two molecules: glycerol carbonate and butyl acetate. The enzyme employed for such reactions is a lipase, a triacylglycerol acylhydrolase (EC 3.1.1.3). These developments are being carried out in partnership with Universidade Federal do Rio de Janeiro (UFRJ) and Universidade do Estado do Rio de Janeiro (UERJ) and have resulted in two patent applications to date.

10.3.1 Glycerol Carbonate

Glycerol carbonate (3) is useful for several and diverse applications, including as a component of gas separation membranes, a surfactant component, a non-volatile component in the paint industry, solvent for other applications, component in coatings, component of detergents and component of new polymeric materials (e.g. hyperbranched aliphatic polyethers).^43,44 The classic route to synthesize glycerol carbonate is to react glycerol (1) with excess dimethyl carbonate (DMC, 2) (Scheme 10.1). For this process, enzymes are very efficient catalysts (a quantitative yield is often achieved) and, in addition, operate at mild temperature and pressure conditions and in solvent-free systems.⁴⁵ The use of immobilized enzymes – which can be easily reused – also offers advantages to this technology towards economic feasibility.^43,45

Scheme 10.1 Classical route of glycerol carbonate (3) synthesis, from glycerol (1) and dimethyl carbonate (2).

Although the use of glycerol, which is a byproduct from biodiesel production, is very attractive, another possible approach to synthesize glycerol carbonate is to react directly vegetable oils with dimethyl carbonate. This route is claimed to be beneficial since it delivers glycerol progressively, thus avoiding inhibition of enzymes.⁴⁶ PETROBRAS development is following this path. Recently, we succeeded in the immobilization of Candida antarctica lipase B (CALB) in Accurel1000 under continuous flow conditions, in a 5 mL packed-bed column. A gradient in the immobilization efficiency between the upper, middle and lower layers was observed and an efficiency of up to 93% was achieved. The immobilized enzyme was subsequently used for glycerol carbonate synthesis by both the classic route from DMC and glycerol and by a proprietary route directly from transesterification of glycerides from acid oils (e.g. palm oil, macauba oil) with DMC.⁴⁷ Selectivity was over 99% in all cases and conversion of up to 99% was achieved at short residence time (176 min), under continuous reaction.⁴⁸ Future developments include extensive tests to determine enzyme stability under the reaction conditions and scale up of the process.

10.3.2 Butyl Acetate

Butyl acetate is one of the short-chain esters with highly relevant industrial applications. It finds use in paints & coatings, pharmaceuticals, plasticizers, electricals and perfumery & flavors (it presents apple and pineapple notes),^49,50 and for end-use mainly in automotive and homebuilding components.⁵¹ The use of biocatalysts for the synthesis of butyl acetate is regarded as promising, especially for application as a flavor (in food, cosmetics and pharmaceutical) due the more environmentally-friendly nature of this route.⁴⁹

Enzyme sources already investigated for the synthesis of butyl acetate are the yeasts Candida antarctica^49,52,53 and Candida rugosa⁵⁴ and the filamentous fungi Rhizopus oryzae⁵⁵ and Rhizomucor miehei.^50,56 The classical reaction studied is between acetic acid and n-butanol;^50,54,56 alternative reaction systems, such as low-frequency ultrasound, have been described more recently as effective in productivity improvement.^49,53

PETROBRAS’ efforts on butyl acetate synthesis have consisted of the use of immobilized CALB in a variety of routes. Different acetic acid concentrations, acyl acceptors (acetic acid, ethyl acetate, acetic anhydride), reagents proportion and the use of solvents have been investigated. Laboratory-scale results (up to 50 mL) indicated up to 96% conversion after 4 h reaction at 50 °C, in the presence of hexane, using the commercial product Novozym® 435 as biocatalyst.⁵⁷ Future developments are to include tests to determine enzyme stability over long reaction times and scale up of the process.

10.4 Synthesis and Degradation of Polymers

10.4.1 Synthesis of Polyesters

Enzymes can be used in the synthesis of a wide variety of polymers, such as polyesters, polyurethanes, polycarbonates, polyamides and polystyrene.⁵⁸ They present many advantages over chemical catalysts, such as exquisite selectivity and control of the polymer structure and physical properties, safer synthesis due to milder operational conditions, absence of residual metal catalyst in the product and good chemo-, regio- and/or stereoselectivities.^59–61 This opens up new end-uses for the synthesized polymers, including in high value-added specialty polymers for biomedical applications.⁶⁰

The main routes for the enzyme-catalyzed synthesis of polyesters are the following repeated ester bond-formation reactions: polycondensation (or condensation polymerization) and ring opening polymerization (ROP), which use bifunctional molecules as monomers. The first occurs mainly by reacting diols (e.g. 1,3-propanediol, 1,4-butanediol, 1,8-octanediol) and dicarboxylic acids (e.g. succinic acid, adipic acid, itaconic acid, sebacic acid) or their corresponding esters,^60,62via dehydration or alcoholysis reactions, although it can arise also from hydroxyacids (e.g. ricinoleic acid, glycolic acid, lactic acid) or mercaptoalkanoic acids (e.g. 11-mercaptoundecanoic acid) and their esters.⁶³ The ROP route employs cyclic esters, such as lactones (e.g. lactide, ε-caprolactone), as monomers,⁶⁰ and generally gives polyesters with higher molecular weight and lower polydispersity.⁶¹

PETROBRAS efforts on synthesis of polyesters, carried out in partnership with UERJ, focused on the polycondensation route between biobased dicarboxylic acids and diols. The use of the biocatalyst Novozym® 435 for the synthesis of poly(butyl sebacate) (4, Scheme 10.2a) and poly(butyl adipate) (7, Scheme 10.2b) resulted in 94% and 91% acid consumption after 48 h reaction at 90 °C and the increase in enzyme loading from 5 to 15 wt% led to an increase in polymer number average molecular weight (M_n) from 3786 to 4916 Da, respectively.⁶⁴

Scheme 10.2 Reactions for the synthesis of (a) poly(butyl sebacate) (4) from sebacic acid (5) and 1,4-butanediol (6); (b) synthesis of poly(butyl adipate) (7) from adipic acid (8) and 1,4-butanediol.

10.4.2 Depolymerization of Poly(ethylene terephthalate)

Poly(ethylene terephthalate) (PET, 13) is one of the most versatile and consumed plastics in the world, with an annual production of over 50 million tons.⁶⁵ It is synthesized starting from the monomers terephthalic acid (TPA, 9) and (mono)ethylene glycol (EG, 10) in one or two esterification steps to sequentially form bis(hydroxyethyl)terephthalate (BHET, 12) and oligomers with a degree of polymerization of up to seven (Scheme 10.3). These molecules are then reacted via polymerization condensation mechanisms (prepolymerization and continuous polymerization steps) to form the polymer, releasing EG from each new linkage formed. Higher molecular weight PET (150–200 repeating units) can be obtained after a solid-state polymerization (SSP) step in which pellets are heated at up to 240 °C in an inert atmosphere.⁶⁶

Scheme 10.3 Hydrolysis reaction of PET (13) depolymerization. BHET (12) and MHET (11) are intermediates and TPA (9) and EG (10) are final products. For most enzymes, step c is much slower than step b.

PET has been for decades regarded as a non-biodegradable compound, but more recent studies indicated that it or its copolymers (e.g. PET containing 5-nitroisophthalic or nitroterephthalic units)⁶⁷ can be in vitro or in vivo depolymerized by the action of hydrolytic enzymes, including lipases,^68,69 cutinases,^69–71 serine esterases⁶⁹ and p-nitrobenzylesterases,⁷² which catalyze the hydrolysis of the ester bonds in the polymer chain. However, most enzymes are seriously inhibited by one of the hydrolysis end-products, mono(hydroxyethyl)terephthalate (MHET, 11)^70,73 and only a few enzymes can efficiently convert it into TPA and EG.^74,75 Some studies differentiate the enzymes responsible for each step of PET hydrolysis (Scheme 10.3), with one acting on PET depolymerization previously reported as PETase and the enzyme that succeeds in catalyzing MHET conversion into the monomers named MHETase.⁷³

The extension of PET depolymerization is strongly dependent on the capability of the enzyme to catalyze such a reaction. From the broad group of esterases and related enzymes, only few biocatalysts have been described so far as efficient for PET breakdown. This is related to structural and catalytic properties of the enzymes and may also be related to PET properties, such as molar mass and crystallinity.⁷⁶ This is corroborated by some studies that comment on the role of accessory proteins (such as hydrofobins) and binding domains (as carbohydrate binding domains – CBMs), with the aim to tune the sorption characteristics to crystalline regions in the polymer structure.^77–80 These factors result in a wide diversity of biotechnological PET depolymerization processes, ranging from a day or few days^73–75 (usually for in vitro hydrolysis) to months^81,82 (mostly reported for in vivo biodegradation).

PETROBRAS’ efforts on PET depolymerization via enzyme-catalyzed reactions started in 2013 and consist of either the use of commercial off-the-shelf biocatalysts or the screening of potential enzyme sources for this process. This research has currently resulted in two patent applications (final numbers not available yet).

The investigation of eleven enzymes for BHET hydrolysis revealed that only CALB could very efficiently convert MHET into TPA and EG, whereas the others catalyzed this step at a much lower reaction rate. On the other hand, CALB is not effective as a PETase, whilst PET depolymerization was shown to be technically feasible with a commercial Humicola insolens cutinase (HiC). This latter enzyme, however, catalyzes the hydrolysis of MHET slowly, so that mole fractions (considering the three final aromatic products from PET depolymerization – BHET, MHET and TPA) for MHET of up to 0.65 were found at early stages of PET hydrolysis. The complementary and synergistic activities of these enzymes were then investigated and the results showed a boosting effect for TPA release when these enzymes were combined in two different proportions (Figure 10.3).⁷⁵

Figure 10.3 Synergy between two enzymatic preparations after 14 days PET bottle hydrolysis at 60 °C.

10.5 Synthesis of Biolubricants

Base oils are the higher proportion constituents in lubricants formulations and are obtained, in general, from petroleum processing. They can be primarily classified as minerals or synthetics, depending on the source or their production process.⁸³ In several lubricant applications, the required increasingly stricter quality standards (viscosity grades, oxidation resistance, pour point, etc.) may not always be reached by conventional mineral oils. Thus, alternative routes to produce base oils have been developed for obtaining products with higher durability and lower environmental impact.

A biolubricant is a biodegradable lubricant capable of being decomposed by microbial action within a determined time period. In general, biodegradability means that a lubricant will be metabolized by microorganisms within 1 year.⁸⁴ Biolubricants are in general obtained from vegetable oils (Figure 10.4). They are used in applications where there is an environmental risk of leaks. The world finished lubricants market is about 35 000 000 tons per year⁸⁵ and biolubricants represent approximately 1% of this total.⁸⁴ Unlike the mineral based finished lubricants market, which has stagnated, the biolubricants market has been growing on average at 10% per year within the last ten years.⁸⁶

Figure 10.4 Structures of the most commonly used esters as biolubricants: trimethylolpropane ester (14), pentaerythritol ester (15) and neopentylglycol ester (16).

Lubricants must present certain physico-chemical properties within determined specifications, such as viscosity, acid number, corrosion, pour point, etc. For biolubricants, since they are in general composed of organic esters, oxidative, thermal and hydrolytic stabilities are also very important. However, as a high oxidation resistant molecule is synthesized, with the double bonds being removed or protected by steric hindrance, one must take into account that the biodegradation by microorganism action will also be affected. Thus, the biggest challenge to overcome in this area is to synthesize new biolubricants with higher oxidation resistance and good biodegradability, or, in other words, to find a balance.⁸⁷

The environmentally friendly nature of vegetable based lubricants and their market benefits have generated the involvement of a large number of companies in this market. For example, Mobil Chemical has implemented a clean line of lubricants production as part of the program Agriculture for Chemistry and Energy (AGRICE). Shell and British Petroleum have signed an agreement with SNCF, a French train company, to develop biodegradable lubricants. In 2002, the Western Europe total lubricants market was 5 020 000 tons per year, of which 50 000 tons per year was based on vegetable oils. On the other hand, the North American lubricants market was 8 250 000 tons per year of which only 25 000 tons per year are vegetable oil based.⁸⁸

In the lubricants industry, lipases have been gradually applied, mainly for synthetic reactions, such as esterification and transesterification. Several factors influence the conversions of an enzymatic transesterification, including used substrate (vegetable oil or alcohol), molar ratio between the substrates, water content in the reaction medium, presence of solvents, temperature, form of enzyme (powder or immobilized), lipase concentration, and so on. Although some literature reports describe biolubricant synthesis using different lipases, it is hard to state any generalization about the optimum reaction conditions, since lipases from different sources tend to respond differently to changes in the reaction medium.⁸⁹

The application of lipases in ester synthesis from trimethylolpropane (TMP) and rapeseed oil was studied by Linko et al.⁹⁰ The transesterification for the synthesis of tri-esters of TMP from rapeseed oil fatty acids was performed at atmospheric pressure in closed or open 13 mL test tubes, and also at reduced pressure (2.0–13.3 kPa). After the reaction, a sample was extracted with acetone and the precipitated enzyme was removed by centrifugation. Conversions greater than 95% in 24 h were observed.

In the transesterification reaction between TMP and rapeseed methyl ester (RMe)⁹¹ the immobilized commercial lipase from Rhizomucor miehei, Lipozyme® IM 20, was initially used, resulting in TMP ester conversion of about 90%, without water addition. Then, the powder lipase from Candida rugosa without any additional organic solvent was investigated. The absence of solvent allows higher substrate and product concentrations, simplifies the post-reaction processes and increases operational safety. At 47 °C and 15% added water, a conversion in TMP tri-ester of 75% in 24 h and of 98% in 68 h was observed. Under these conditions, by-product formation did not occur and also no residual RMe was observed.

Based on previous studies⁹² that clearly showed that RMe conversion in TMP esters can be increased by using an immobilized lipase, and several support materials for Candida rugosa immobilization were investigated. The highest conversion (about 95%) was achieved with lipase immobilized on Celite® R-630. Other supports such as Duolites ES-561 and ES-762, GDC 200, GCC and HPA 25 gave conversions of approximately 70%. Commercial immobilized lipase Rhizomucor miehei Lipozyme IM 20 gave a conversion of 90%.

In summary, there is plenty of literature evidence about the feasibility of using lipases as biocatalysts for ester synthesis from vegetable oils for biodiesel and for biolubricants applications. Works at PETROBRAS in cooperation with UFRJ resulted in novel use of the Candida rugosa lipase in biolubricant synthesis reactions derived from castor oil (Scheme 10.4).⁹³ Biolubricants were produced in high yields and showed excellent properties, leading to a patent application.⁹⁴ In this invention, the enzymatic catalyzed production of synthetic base lubricants (19), from castor (methyl ricinoleate, 17), soybean and Jatropha methyl biodiesels as raw materials, was investigated.

Scheme 10.4 Transesterification enzymatic reaction for biolubricant production from methyl ricinoleate derived from castor oil: methyl ricinoleate (17), trimethylolpropane (18), methanol and biolubricant (trimethylolpropyl ricinoleate) (19).

The influence of process variables (enzyme type and concentration, water concentration, temperature, biodiesel concentration, reaction time) was studied. Products and substrates conversions of the transesterification reactions were determined by HPLC. Temperature was the most significant variable on the percent conversion. The best conversion was greater than 95%, after 24 h of reaction, using Candida rugosa lipase as biocatalyst. The products had the following properties: viscosity, 290.2 cSt at 40 °C and 28.46 cSt at 100 °C; viscosity index, 132; pour point, −39 °C; and RPVOT (oxidative stability), 42 min. Such properties qualify the product for potential biolubricant applications, in industrial or marine formulations. Among the investigated raw materials, castor biodiesel had the most promising physico-chemical properties. The usage of castor biodiesel to produce biolubricants can be very profitable.⁹³ Based on the promising bench-scale results, PETROBRAS has tried to reproduce this enzymatic process on a pilot scale, to obtain information for an economic assessment. Since it is a batch reaction process, this scale up would not be so difficult. However, due to the usage of lyophilized lipase, some clogging problems were faced. Then, PETROBRAS and UFRJ started a new cooperation project to produce suitable immobilized lipases for their batch biolubricant production process. Only with the successful scale up of such a process will they be able to perform a realistic economic assessment of it.

Vegetable oils are suitable as raw materials for the synthesis of biolubricants for applications in which operation temperatures are lower than 120 °C. On the other hand, vegetable oils have poorer low temperature properties than synthetic lubricants, mineral oils and chemically modified mineral oils (CMMOs). Many of these lubricants show excellent low temperature properties and can be used in Arctic conditions for long periods of time. However, vegetable oils must be used in applications where the process temperature is higher than −40 °C.⁹⁵

Lubricants may be classified in two main categories: automotive and industrial. Automotive oils make up more than 70% of the total lubricants market volume, and the rest are grouped as industrial lubricants. Due to their higher cost, the best applications for vegetable based lubricants are those where their environmental advantages may be maximized.⁹⁵

Low temperature viscosity is one of the most important properties of modern engine lubricants. Cold cranking causes engine wear, and can be overcome with the use of products that produce an immediate effective lubrication. To meet these specifications related to energy efficiency, low viscosity and low evaporation oils have been introduced into the market. Transesterified vegetable oils are good candidates to be used as engine oil due to their superior thermal stability compared to vegetable oils. They also have low viscosity, low deposit formation (which leads to longer oil changing intervals and cleaner systems) and better low temperature properties. Vegetable oils show some disadvantages for engine oil application, such as increasing viscosity during usage, because of its oxidation; shorter oil change intervals; incompatibility with mineral oils, which requires purging the engine before oil changing; and limitations at low temperature for particular formulations. Engine manufacturers are not inclined to accept engine oil that requires a short oil changing interval.⁹⁵

Several recent patents and publications have reported the technical feasibility of the enzymatic process for biolubricants (esters) synthesis, using lipases as biocatalyst. In some cases, enzymatic process has resulted in higher conversions (approx. 98%) than chemical process (approx. 60%), for the same reactions.^93,96

10.6 Synthesis of Biodiesel

Biodiesel is a biofuel consisting of fatty acid monoalkyl esters (FAAEs) that presents similar properties to fossil-based diesel with the advantage of lower emissions of particulates, CO, SO_x and aromatic hydrocarbons.⁹⁷ It is conventionally synthesized via alkaline or acid chemical catalysis, but the use of enzymes as catalysts has been increasingly studied (Figure 10.5) due some advantages of this route, which include environmentally-friendly aspect, possibility to use low quality/low cost raw materials (e.g. high acidity oils), possibility of having simultaneous esterification and transesterification reactions, easier recovery and better quality of glycerin and operation at mild conditions.^97,98 Enzyme-catalyzed synthesis of biodiesel can occur via two primary routes (Scheme 10.5):

Hydroesterification:^99,100 this consists of a first step of glycerides hydrolysis generally followed by glycerol separation, and a second step of FAEE synthesis (esterification) by reacting free fatty acids with a short chain alcohol (generally methanol or ethanol).
Transesterification:^99,101 this route corresponds to a one-pot FAEE synthesis from the primary raw material, via alcoholysis reactions, generally with a short-chain alcohol.

Figure 10.5 Scientific documents reporting enzyme-catalyzed biodiesel synthesis; 2016 are partial data. Source: Scopus. www.scopus.com. Search on 6 October 2016.

Scheme 10.5 Reactions employed for the synthesis of biodiesel.

The efficiency of enzyme-catalyzed biodiesel synthesis is affected by several environmental factors, such as specificity of the enzyme employed, temperature, water content, type of alcohol, alcohol concentration, presence or absence of solvent (and type of solvent), biocatalyst loading and agitation speed.¹⁰² In addition, the type of bioreactor (stirred tank or column) and mode of operation (batch, fed-batch, batch with recirculation, continuous) influence process performance.⁹⁸ Therefore, process optimization for each specific set of variables is necessary to achieve an industrially feasible technology.

PETROBRAS’ efforts on biocatalyzed biodiesel production consist of a variety of enzyme sources and routes. The research was carried out in cooperation with some universities, including UFRJ, UERJ, Universidade de Campinas – Unicamp and URI Erechim, and resulted in at least six patent applications. The enzymes already investigated are produced by filamentous fungi (e.g. Aspergillus parasiticus, Penicillium simplicissimum, P. verrucosum, Rhizomucor miehei),^97,103–106 yeasts (Candida antarctica, Yarrowia lipolytica)^107,108 and plants (e.g. Castor bean).⁹⁷ Enzyme production was studied, mostly by solidstate fermentation (SSF) of agroindustrial byproducts and wastes, such as babassu (Orbygnya oleifera) cake,^97,106 castor bean cake,^103,104,109 cotton (Helianthus annuus) seed and soybean (Glycine max) meals,¹⁰⁷Jatropha curcas cake¹⁰⁶ and macauba (Acrocomia aculeata) cake;¹¹⁰ submerged fermentation of residual glycerol from biodiesel production has also been investigated.¹⁰⁸ Lipase production was investigated at a scale of up to 1 kg under solid-state fermentation with filamentous fungi and of up to 100 L under submerged fermentation with recombinant Pichia pastoris expressing CALB gene. In submerged fermentation, the observed productivity was 14% higher than for enzyme production carried out in 5 L bioreactor (unpublished work).

The studies also included methods for improvement of enzyme activity and stability (e.g. via immobilization on selected supports)^109,111,112 and the use of dry fermented solids from SSF containing lipase activity as biocatalyst.⁹⁷

In a project in collaboration with PETROBRAS, Aguieiras et al.⁹⁷ investigated at laboratory scale an enzyme–enzyme hydroesterification process for biodiesel production from acid (10.5% acidity) macauba oil, employing a vegetable enzyme obtained from dormant castor seeds in the hydrolysis step and a fermented solid obtained from SSF of babassu cake by Rhizomucor miehei in the synthesis step. The conversions achieved after the first and the second steps were 99.6% and 91%, after 6 h and 8 h reaction, respectively. The fermented solid used as biocatalyst in the second step could be reused for eight sequential cycles with retention of at least 60% of its initial activity.

In another project in collaboration with PETROBRAS, Cavalcanti-Oliveira et al.¹¹³ identified different lipase-producing microorganisms that infected macauba fruits, probably being responsible for the acidity increase of oil, which led to its disqualification for direct use in conventional transesterification technologies for biodiesel production. This observation elucidated the origin of oil acidification and allowed the proposal of proper methods to prevent this phenomenon.

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108. A. M. Castro, J. V. Bevilaqua, D. M. G. Freire, F. A. G. Torres, L. M. M. Santa Anna, M. L. E. Gutarra, C. A. Barbosa, R. V. Almeida, R. R. Menezes and A. G. Cunha, US Pat. Appl. US 2011/0183400 A1, 2011.

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10.1 PETROBRAS Overview