Lipase immobilized graphene oxide biocatalyst assisted enzymatic transesterification of Pongamia pinnata (Karanja) oil and downstream enrichment of biodiesel by solar-driven direct contact membrane distillation followed by ultrafiltration

https://doi.org/10.1016/j.fuproc.2020.106577Get rights and content

Highlights

  • Efficiency of membrane-integrated enzymatic transesterification evaluated

  • Biocatalyst developed efficiently via lipase immobilization onto GO by adsorption

  • It showed better stability, selectivity and catalytic activity with ~88% FAAE yield

  • Solar-driven DCMD followed by UF system ensures high purity of biodiesel

  • Energy-efficient and eco-friendly system represent sustainable biofuel production

Abstract

The present experimental investigation has shown the biodiesel production from 2nd generation renewable carbon source, Karanja seed oil using graphene oxide (GO) immobilized lipase biocatalyst. The adsorption of lipase in GO was found to be most suitable immobilizing support matrix (adsorbed ~94 mg/g of GO) due to large specific surface area and abundant functional groups. The GO immobilized enzyme i.e. biocatalysts assisted enzymatic transesterification yielded ~88% of biodiesel using 1:8 Karanja oil: ethanol ratio, 25 °C, 3% biocatalysts in 24 h reaction time. The newly designed solar-driven direct contact membrane distillation module enables continuous separation and recycles of unreacted alcohol (>99% recovery) from transesterification reaction mixture while yielding ~41 Kg EtOH/m2/24h using commercial PTFE/PET hydrophobic membrane. Subsequently, free glycerol was removed up to ~77% (permeate concentration ≤0.018 mass%) by cross-flow membrane module fitted with flat-sheet polyethersulfone ultrafiltration membrane. The integration of GO-based biocatalyst assisted transesterification and multi-staged membrane system for downstream enrichment of fatty acid ethyl ester culminates in the development of a cost-effective, energy-intensive and eco-friendly system which represents a sustainable biofuel production process.

Introduction

The increasing accumulation of greenhouse gases (GHGs) in the atmosphere and rapid consumption of fossil fuels have impacted climate change seriously as well as depleted the fossil fuels reserves. This leads to exploration into alternative, the renewable and clean energy source to minimize the dependency on fossil fuels as well as reducing the GHGs emission [1]. Biodiesel, being a promising substitute to conventional fuels, is a mixture of fatty acid alkyl esters (FAAE) obtained from the catalytic transesterification of plant or animal oils and alcohols. Generally, chemical and enzymatic catalysts are used for biodiesel production through catalytic route [2]. Chemical catalysts, despite being faster and cost-effective suffer limitations such as low product purity, high energy consumption, sensitive to the quality of feedstocks, generation of wastewater and soap formation [3,4]. More attention has been paid towards the development of heterogeneous biocatalyst such as enzymatic transesterification of triglyceride feedstock which is found very effective in yielding pure biodiesel through the elimination of any side products [5].

Mild process conditions, little or no side reaction, easy downstream purification, minimal waste generation, less energy consumption and relatively less or no pretreatment of feedstocks are offered by enzymatic route of transesterification that leads to an economic and cheaper product. Also, the enzymatic method ensures high quality of the final product in the absence of a saponification process and suitable for a broad variety of oil due to resistant to higher FFA content in the feedstocks [6]. One of the most useful and eco-friendly biocatalysts employed for transesterification is lipase, which is hydrophobic proteins that act on carboxylic acid esters like glyceride lipids at the interface of aqueous and oil, giving high conversion yield of biodiesel [7]. Despite several advantages, enzymes are hardly competitive in terms of cost with chemical catalysts-based biodiesel synthesis [8]. Moreover, the main challenge in the widespread use of enzyme is its instability at high temperature or harsh solvents and inability of enzyme recovery [9]. However, immobilization of enzyme has shown better resistant to thermal and solvent exposure retains its catalytic activities at extreme conditions, easily separate from the product stream, amenable for prolonged use and continuous operation to counterbalance the high cost [10]. Different support materials have been proposed as a carrier for immobilization to improve the thermal and pH stability of the lipase activity such as carbonaceous supports, silica-based carriers and synthetic polymers [11,12]. More recently, magnetic responsive nanoparticles as magnetic composites like poly(styrene-methacrylic acid) microspheres [13], ionic liquid-functionalized magnetic silica [14], Fe3O4-poly (glycidyl methacrylate-co-methacrylic acid) [15] were used to covalently entrapped the lipase for easy biocatalyst recovery using an external magnetic field. However, aggregation of magnetic nanoparticles in liquid reaction mixture under strong magneto-dipole interactions and large surface energy could hamper and reduce the catalytic efficiency of the enzyme [16]. Moreover, the high cost of such support materials causes many to search for cheaper, higher surface area and better stability material.

One of the alternatives and very less investigated, graphene oxide (GO) appears to be most attractive since the GO-based supports are used successfully for various biologically active agents such as novel biocatalysts, biosensor and drug delivery vehicles [17]. To reduce the up-stream operating cost, immobilization of the catalytic stability and activity of lipase can be done by adsorption onto porous support materials like GO which provide large surface area, low diffusional resistance and high enzyme loading capability. Several other distinctive characteristics of GO such as biodegradability, antioxidant properties, thermal stability and high pore volume make it a suitable candidate for biomolecules/enzymes immobilization [18]. This enables better interaction with enzyme, higher efficiency of immobilization and increase in long-term storage as well as enhance its lifetime in transesterification. Besides, immobilized biocatalyst provide substantial benefits viz. process simplification, reduced carbon footprint and more sustainable process with promising industrial-scale implementation compared to chemical catalysts [19].

The lipase catalytic mechanism displays two conformational forms during its action viz. a closed-form where a lid of polypeptide chain hides the active centre from the medium and an open-form where the active centre is exposed to the reaction medium due to displacement of lid [20]. The active centre of the lipase adsorbed smoothly to the large hydrophobic pockets on the GO surfaces and the open-form stabilizes through the phenomenon of interfacial activation which improves the activity, selectivity and pureness of lipase [21]. Thus, supporting morphological structure, large surface area and formation of a stable aqueous suspension, the GO nano-sheet have shown the ideal candidate for better enzyme loading and support material [10]. Active oxygen-containing functional groups are present on GO surface which help to immobilize enzyme using covalent bonding and/or non-covalent bonding such as weak interactions without any surface modification or any coupling reagents [22]. Thus, nanostructured GO sheet, enriched with the oxygen-containing groups may be considered as the ideal substrate for the study of enzyme immobilization for different applications [23]. There are only a few studies on the immobilization of lipase in GO and used as a biocatalyst for transesterification reaction [12,24].

The economics of transesterification is mainly influenced by the cost of feedstock, recycle of catalyst, recovery of unreacted alcohol as well as a by-product (glycerol) and the additional purification steps involves during the downstream separation of FAAE [5]. In most of the studies, methanol is used as an alcohol source due to high yield and efficient production and separation of biodiesel. However, methanol is a petroleum by-product and non-renewable resource which is toxic. On the other hand, bioethanol is obtained from renewable resources which is a good substitute of methanol for the sustainable production of fatty acid ethyl ester (FAEE) biodiesel [25]. Traditionally, downstream separation of the biodiesel either dry or wet washing involves several energy-intensive unit operations such as centrifugation, filtration, decantation, gravitation settling, sedimentation, distillation, adsorption, dehydration and ion-exchange [5]. In wet washing, most of the impurities (Glycerol) are removed but the use of water could increase cost, production time and generate huge quantity of wastewater [26]. Dry-washing is also costly and its chemistry is still not fully understood [27]. Adverse environmental conditions such as high pressure (5000 Pa) and temperature (250 °C) is required to refine the glycerol-free biodiesel through distillation [28]. In literature, most of the studies have shown the recovery of unreacted alcohols (MeOH/EtOH) by using distillation, but despite its widespread use and benefits, a key disadvantage is its high energy requirement [5].

A paradigm shift is now imminent to get the pure and economically viable product in the downstream separation of biodiesel by choosing membrane-based emerging technologies. The purity of biodiesel is an utmost important issue and must follow the international standards such as ASTM D6751 (USA) and EN14214 (Europe) [29]. The main aim is to limit the presence of impurities such as residual alcohol, catalysts, water, and free as well as bound glycerol (e.g. monoglyceride (MG), diglyceride (DG), triglyceride (TG)) that may affect the engine performance because of lower fuel flashpoint, oxidation stability, increase viscosity, form ice crystals, damage rubber seals, and gaskets [5]. Excess free glycerol (>0.02 mass %) content in biofuels can create problem during storage and decantation because of gum-like deposits around injector tips and valve head [30]. Membrane-based separation and purification technology can be successfully applied for transforming the conventional production plant to a compact, energy-saving, flexible, economical, eco-friendly with an enhanced capacity [31]. The membranes are being used as an effective approach in the form of cross-flow membrane unit, membrane contractor, membrane adsorption, membrane distillation (MD), membrane pervaporation as partial or full in chemical production processes. The same approach has been well investigated during downstream purification of biodiesel focusing on size exclusion of TG, DG and MG using carbon membrane (pore size 0.05 μm) from MeOH, Gly and FAAE [32]. The ultrafiltration (UF) ceramic membrane (0.14 μm) was applied to separate alcohol rich phase (methanol and FAAE) from the ester rich phase (oil and FAAE) using a simulated mixture of oil/MeOH/biodiesel [33]. However, ceramic membranes have less contact area in comparison to polymeric membranes as well as more expensive. The unreacted alcohol can be recovered and reused by using solar energy-driven MD process using hydrophobic membrane from the crude biodiesel mixture [34].

In the present investigation, a novel GO immobilized lipase biocatalyst was developed for enzymatic transesterification of Karanja oil. The study is focused on the application of different membrane systems for stepwise downstream separation of valuable ingredients from the crude biodiesel mixture after transesterification. The application of two-step membrane separation technology viz. solar-driven direct contact membrane distillation (DCMD) and UF system have been evaluated for the separation as well as recovery of the unreacted alcohol and purification FAAE from glycerol. Thus, application of multi-staged membrane system along with biocatalyst for the production, separation and purification of renewable energy source (biodiesel) using cheap and easily available non-edible plant oil represents compact, flexible, eco-friendly plant leading to the prospect of process intensification in the backdrop of absence of similar study in the literature.

Section snippets

Reagents and membranes

The analytical grade of chemicals was purchased in the present investigation. For the preparation of GO matrix, graphite powder (<20 μm), H2SO4 (98%), HNO3 (98%), H2O2 (30%), Na2NO3, KMnO4, HCl (35%), acetone (99.9%) and isopropanol (99.9%) were procured from Sigma-Aldrich, Merck India Pvt. Ltd., Sisco Research Laboratory (SRL), India. Other chemicals such as NaOH pallets, NaCl, CH3COONa, KH2PO4, K2HPO4, ethanol (99.5%), glycerol (99.5%) and the lipase (Steapsin, activity ~40–70 U/mg) ex.

Characterization of biocatalyst

The oxygen-containing functional groups in a honeycomb lattice of GO structure has numerous advantages as an immobilizing matrix for the functional materials such as enzymes, nanoparticles, and organic compounds. Thus GO nanosheet can be used as a host grafting material to board biomolecules due to the vast specific surface area, stable structure, staggered, and plentiful functional groups. The specific surface area and pore volume of GO were measured by BET equation using N2 gas

Conclusions

Enzyme immobilized GO-based biocatalyst was developed and tested in enzymatic transesterification of Karanja oil. The GO nanosheets-based support material has effectively adsorbed the lipase with the loading of 94 mg/g in a phosphate buffer solution of pH 6.5 towards maximization of catalytic activity in transesterification reaction by giving 88% of the biodiesel yield. Two-steps membrane-based downstream purification processes (viz. DCMD and UF) have been introduced to recover >99% residual

CRediT authorship contribution statement

Ramesh Kumar: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft, Writing - review & editing.

Parimal Pal: Conceptualization, Funding acquisition, Formal analysis, Providing infrastructure/resources, review writing, editing.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Acknowledgements

The author gratefully acknowledges to the University Grant Commission, New Delhi, India for providing fund under the scheme of Dr. D.S. Kothari PDF, Sanctioned No. F.4-2/2006 (BSR)/EN/16-17/0001.

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