Use of a low-cost methodology for biodetoxification of castor bean waste and lipase production

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Abstract

The castor bean (Ricinus communis) represents a potential candidate for biodiesel production. The Petrobras Research Center is developing a biodiesel production process from castor bean seeds, in which an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste is toxic and allergenic and, as such, poses a significant environmental problem. Solid-state fermentation (SSF) of castor bean waste was carried out to achieve ricin detoxification, reduce allergenic potential and stimulate lipase production. The fungus, Penicillium simplicissimum, an excellent lipase producer, was able to grow and produce lipase enzyme. After an optimization process, the maximum lipase activity achieved was 44.8 U/g. Moreover, the fungus P. simplicissimum was able to reduce the ricin content to non-detectable levels in addition to diminishing castor bean waste allergenic potential by approximately 16%. In this way, SSF of castor bean waste by P. simplicissimum may increase the utility of the waste by promoting enzyme production and eliminating the principal toxic element, ricin.

Introduction

The castor bean consists of approximately 50% oil, giving it special characteristics such as high viscosity, heat and pressure stability, low freezing point, and the ability to form waxy substances after chemical treatments [1]. Consequently, the castor bean is a promising candidate for biodiesel production. In recent years, the Petrobras Research Center has been developing a biodiesel production process from castor bean seeds [2]. After a transesterification reaction, an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste presents no utility after the biodiesel production process and it cannot be used as animal feed. Furthermore, castor bean seeds contain a potent toxin (ricin) and an allergenic protein fraction (CB-1A or 2S albumin isoforms), which severely limit the utility of castor bean waste after oil extraction [3], [4]. The ricin toxin is a 62–66 kDa protein produced by castor beans (Ricinus communis). This holotoxin consists of two polypeptide chains, approximately 32 kDa and 34 kDa in size, linked by a disulfide bond. The A chain (RTA) is a potent ribotoxin, inhibiting protein synthesis in mammalian cells at doses as low as a single RTA molecule per cell. The B chain (RTB) is a lectin, which binds to galactose residues on the cell surface. The estimated lethal ricin dose in humans is 1–10 μg/kg [3], [5]. The allergenic compounds of castor bean waste are stable proteins that exhibit an extraordinary capacity to sensitize individuals exposed to low concentrations of castor bean powder or castor bean waste. Once industrial-scale production of biodiesel from castor bean is established, a great amount of the waste will inevitably be produced. The total elimination or inactivation of toxic compounds from castor bean waste is extremely important before it can be considered useful as animal feed, fertilizer or in wastewater pre-treatment (as solid enzymatic preparation). Even if its final destination is the landfill, it is necessary to eliminate the waste's toxicity and avoid contamination of the earth's soil and waters.

Attempts have been made to detoxify the castor cake, the byproduct of castor oil extraction, using physical and chemical methods [6]. Biological detoxification, using solid-state fermentation (SSF) with filamentous fungi, has been used to detoxify other residues, showing good results [7]. In addition to promoting residue-detoxification, SSF of castor bean waste represents an interesting and low-cost alternative for generating useful enzymes, such as lipases [8], [9].

Lipases (E.C. 3.1.1.3) are carboxyl esterases that catalyze the hydrolysis of acylglycerols containing fatty acid chains greater than 10 carbon atoms in length [10]. Lipases can also catalyze reverse reactions of fatty acid breakdown, such as esterification and transesterification, in non-aqueous environments. These enzymes also demonstrate regio- and enantioselectivity. Due to these characteristics, lipases possess a wide range of application in industrial and fine chemicals applications [11]. However, as in any application that demands high quantities of enzyme, such as biodiesel production or wastewater treatment, the cost of lipase production must be reduced in order become economically viable.

The aim of this work was to study the biological detoxification of castor bean waste and, simultaneously, attempt to add value to the waste residue through an acidic thermo-stable lipase production [12] in SSF using Penicillium simplicissimum—a strain previously shown to be an excellent lipase producer [13]. Further, the biodetoxification process described here can extend the use of fermented castor bean waste and potentially be used as animal feed or fertilizer, without causing damage to the environment.

Section snippets

Microorganism and culture mediums

The P. simplicissimum strain was isolated and selected [13] as a promising lipase producer in solid-state fermentation. To obtain spores, the fungal strain was propagated at 30 °C for 7 days in a medium containing the following supplements (% w/v): 2.0 soluble starch; 0.025 MgSO4·7H2O; 0.05 KH2PO4; 0.5 CaCO3; 0.1 yeast extract; 1.0 olive oil; and 1.0 agar. The spores were suspended in phosphate buffer (100 mM, pH 7) and counted in a cell-counting chamber.

Solid-state fermentation

Castor bean waste, a solid biodiesel

P. simplicissimum lipase production in castor bean waste

The wild Brazilian P. simplicissimum strain was able to grow in castor bean medium by solid-state fermentation and capable of producing an extracellular lipase in lab-scale, tray-type bioreactors. The changes in lipase production, moisture contents, water activity and pH during SSF of castor bean waste are presented in Fig. 1.

Prior to optimizing culture conditions, P. simplicissimum exhibited maximum lipase activity (16.9 U/g), productivity (0.23 U/g h) and specific activity (2.02 U/mg of protein)

Conclusion

The P. simplicissimum strain used was able to grow in SSF of castor bean waste and demonstrate substantial lipase production. The maximum lipase activity reached was 44.8 U/g, about 263% greater when compared to activities reached before the optimization process. Moreover, the fermentation process was able to eliminate the ricin and reduce the allergenic potential of the waste, proving to be an effective method for residue detoxification. Therefore, it was possible to generate value from a

Acknowledgements

This work was supported by CENPES/PETROBRAS and the Brazilian governmental funding agencies FAPERJ, CAPES and CNPq.

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