BR-102024017477-A2 - METHOD FOR OBTAINING A LYOPHILIZED ENZYMATIC PREPARATION AND ITS USES IN SYNTHESIS REACTIONS FOR THE PRODUCTION OF STOLIDE-TYPE BIOLUBRICANTS, BIODIESEL AND POLYOL-ESTER TYPE BIOLUBRICANTS

Abstract

The present invention describes a method for obtaining a suitable biocatalyst, called lyophilized enzymatic preparation (LEP) via submerged fermentation (SF) of rough Diutina yeast. The biocatalyst obtained by the process is also applied in synthesis reactions for the production of estolide-type biolubricants; biodiesel and polyol-ester type biolubricants.

Inventors

• DENISE MARIA GUIMARÃES FREIRE
• ELISA D?AVILA COSTA CAVALCANTI
• CHARLES LIMA BESSA ASSUNCAO
• ANA CRISTINA DE ARAUJO COLLAÇO
• JOSE ANDRE CAVALCANTI DA SILVA
• ERIKA CRISTINA GONÇALVES AGUIEIRAS

Assignees

• Petróleo Brasileiro S.A. - Petrobras
• UNIVERSIDADE FEDERAL DO RIO DE JANEIRO ? UFRJ

Dates

Publication Date

20260310

Application Date

20240826

Claims (12)

1. 1. Method for obtaining a lyophilized enzymatic preparation (LEP) characterized by comprising the following steps: a) submerged fermentation (SF) of rough Diutina yeast; b) centrifugation of the fermented medium, after the end of fermentation, obtaining cells (5) and a supernatant (S); c) lyophilization of the supernatant (S) obtaining the LEP (7) as a biocatalyst.
2. 2. Method, according to claim 1, characterized by the submerged fermentation being in fed-batch mode using oleic acid as a carbon source at a feed rate of 0.43 to 2 g/l/h.
3. 3. Method, according to claims 1 and 2, characterized by submerged fermentation occurring at a temperature of 30°C to 40°C; aeration of 0.5 to 3 vvm; pH of 6 to 7; and agitation of 200 to 600 rpm.
4. 4. Method, according to claims 1, 2 and 3, characterized in that the PEL is not purified and formulated.
5. 5. Use of PEL, obtained by the method as defined in claims 1 to 4, characterized by being in its crude form and in synthesis reactions for the production of estolide-type biolubricants; biodiesel and polyol-ester type biolubricants.
6. 6. Use, according to claim 5, characterized by, in the synthesis reactions for the production of estolide-type biolubricants, using castor oil-free fatty acid (12) as a substrate and PEL (7) as a biocatalyst.
7. 7. Use, according to claim 6, characterized by the synthesis reaction for the production of estolide-type biolubricants occurring under agitation of 200 rpm and a temperature of 35 to 45°C, under atmospheric pressure in a reactor (11).
8. 8. Use, according to claim 5, characterized by, in the synthesis reactions for the production of biodiesel, using distillate from the deodorization of palm oil (DDOP)(15) and hydrated ethanol (95%) (16) as substrates or castor free fatty acid (CFFA) (12) or soy free fatty acid (SFFFA) (22) as substrates and PEL (7) as a biocatalyst.
9. 9. Use, according to claim 8, characterized in that the synthesis reaction for the production of biodiesel occurs under agitation of 200 rpm and a temperature of 40 to 50°C, under atmospheric pressure and at a molar ratio of hydrated ethanol (95%):DDOP of 1:1 in a reactor (11).
10. 10. Use, according to claim 5, characterized by, in the synthesis reactions for the production of polyol-ester type biolubricants, using free fatty acid from soy (AFLS) (22) and polyalcohol (Neopentyl glycol - NPG) (23) as substrate and PEL (7) as biocatalyst.
11. 11. Use, according to claim 10, characterized in that the synthesis reaction for the production of polyol-ester type biolubricants occurs under agitation of 200 rpm and temperature of 35 to 45°C, under atmospheric pressure and at a molar ratio of NPG:AGLS of 1:2.5 in a reactor (11).
12. 12. Use, according to claim 10, characterized in that the polyalcohol (23) is optionally trimethylolpropane (TMP) or pentaerythritol (PET).

Description

Field of invention [01] The present invention has as its field of application industrial plants for the production of biodiesel and biolubricants from vegetable oils. Concomitantly, the present invention is situated in the field of production of enzymatic biocatalysts that are used in synthesis reactions to obtain biodiesel and biolubricants from vegetable oils. Fundamentals of the invention [02] Biolubricants derived from vegetable oils are considered an alternative to the use of petroleum-derived lubricants, since they are biodegradable organic esters, that is, they can be decomposed through microbial metabolism, in addition to having physical and chemical properties similar to lubricants of mineral origin. [03] In the conventional process of producing biolubricants, acidic chemical catalysts, such as sulfuric acid and perchloric acid, are used at high temperatures (200°C) and pressures for the esterification reaction of fatty acids. However, in addition to generating acidic effluents, the use of strong acids can lead to the breakdown of functional groups, releasing sulfonated acid groups that lower the pH, impairing the performance of the process. [04] Another product of industrial interest is biodiesel. Biodiesel is industrially produced from a transesterification reaction, in which methanol reacts with the triglyceride present in vegetable oil by the action of a homogeneous catalyst (usually NaOH and KOH), generating a mixture of fatty acid methyl esters (biodiesel) and one mole of glycerin for each mole of triglyceride. Although the use of homogeneous alkaline catalysts presents high reaction rates, being a well-established and low-cost process, the use of these catalysts presents many disadvantages, such as the difficulty in separating them from the product, the possibility of saponification reactions when using raw materials with a high content of free fatty acids (> 0.5% by mass), high energy consumption, and large quantities of alkaline wastewater. Thus, enzymatic synthesis appears as an interesting alternative for obtaining biolubricants and biodiesel compared to conventional chemical methods. [05] Because enzymes are catalysts with high specificity, low concentrations of reaction byproducts are generated, which reduces the amount of industrial effluents. In addition, the energy consumption of the process is reduced, since enzymes act under mild reaction conditions of temperature, pH and pressure. Considering enzymatic synthesis, the use of proprietary lipases produced by submerged fermentation as proposed by the present invention proves to be an even more promising alternative when compared to the use of high-cost commercial lipases and the use of chemical catalysts. [06] Commercial rough diutin lipase (Lipomod 34 MDP) has been used in biodiesel production reactions, showing good results in terms of ester conversion. [07] As examples, MÉNDEZ et al., (2022), evaluated the production of biodiesel via transesterification of coconut oil, obtaining a biodiesel yield of 98%. In addition, IULIANO et al., (2020), employed rough diutin lipase for the conversion of spent grains oil (SGO) into biodiesel, in the presence of methanol, with a yield of 98%. XIE et al., (2020), evaluated an organic copolymer to be encapsulated in Fe3O4 nanoparticles to which rough diutin lipase was bound. The biodiesel yield was maintained at 79.4% after reuse for five cycles. Furthermore, this lipase has been used to obtain biolubricants from polyols such as trimethylolpropane (TMP), neopentyl glycol (NPG) and pentaerythritol (PET) as substrates. PAPADAKI et al. (2018) used microbial oil from Rhodosporidium toruloides and Cryptococcus curvatus to produce biolubricants with NPG. Conversions greater than 82% were achieved. On the other hand, palm oil deodorization distillate (DDOP) is another substrate that can be used with NPG or TMP, as described by FERNANDES et al. (2018), achieving maximum OH esterification to DDOP-TMP esters (94%) and DDOP-NPG esters (87%) using 4% Lipomod 34 MDP. FERNANDES et al. (2021) evaluated the synthesis of biolubricants from distilled soybean fatty acids (DDOS) using rough diutin lipase in free and immobilized form using NPG and TMP as polyalcohols. Furthermore, recent studies report the use of this enzyme in estolide (biolubricant) synthesis reactions using castor oil-free fatty acid (COFA) as a substrate. GRECO-DUARTE et al. (2017) described that Lipomod 34 MDP exhibits greater specificity for ricinoleic acid compared to polyols. The reduction in acidity in the reaction with COFA was similar to that obtained in the presence of polyols. Two other commercial enzymes, from Rhizomucor miehei and Candida antarctica, were unable to reduce acidity in this reaction medium without polyols. GRECO-DUARTE et al. (2019) also described a kinetic medium for controlling the size of estolides, in which the analysis showed that estolides of various sizes [dimers, trimers, tetramers, and pentamers (+)] were produced