Immobilization microbial cellulase and lipase as bio-catalyst agent using maltodextrin for biofuel synthesis

The alternative method of using enzyme technology and modification methods will reduce the cost of biofuel production. New solution for biofuel production by improving the advantages from potential microbial to produce biocatalyst such as cellulase and lipase as an alternative catalyst for replacing the chemical catalyst for transesterification process. This study was developing new method of enzyme immobilization for feasibility biofuel production which is more efficient and effective process in industries. The result obtained in this work allowed concluding that the highest value for the total cellulolytic activity was obtain using carboxymethyl cellulose as substrate reaching value 4.225 U/ml for 24 h with optimization of pH 7 and temperature 50 °C. Instead of cellulase, the potential Aspergillus niger InaCC F538 can produce extracellular lipase and the value found for lipase activity in olive oil substrate is 3.710 U/ml for 24 h incubation time with optimization of pH 8 and temperature 50 °C. The immobilization process of cellulase and lipase using maltodextrin was enhancing the activity to 19.615 U/ml and 24.123 U/ml. The biofuel production based on microbial will be widely used for continuously used for renewable raw material from waste agriculture biomasses for biofuel production. New enzymes combination for improving enzyme stability for industrial application based on fermentation through the economically feasible.


Introduction
The new green wave will save all the countries in the international communities which are face enormous challenges with regards to the issues of global population, dwindling fossil fuel resources, and ever-advancing climate changes towards global warming.This research has focused on production and immobilized cellulase and lipase as biocatalyst as a bio-agent for synthesis bioethanol and biodiesel for simultaneous and continuing process.Improving potential microbes for producing biocatalyst and making the immobilized enzyme for reuse dan continuously used in bioreactor because of its operational was stable and low cost for creating economically solution for biofuel production.
Cellulolytic activity has been studied by several methods and it shows the summarized of unit per milliliter in recent papers [1,2].Crystalline cellulose is degraded at very slow range and most assays were adapted to use very light degradable soluble cellulose derivatives like carbohydrate substrates such as carboxymethylcellulose (CMC) [3,4].Screening for extracellular cellulase production by microbes such as bacteria and fungi is using general methods like inoculate the microbe onto agar plates containing CMC as substrate [5].The application of cellulase from microorganisms have developed the new technological for cellulase production remain an important issue that enable to developing the sustainable process for ethanol production from agriculture biomasses [6,7].Bioprospection of microorganisms which is potential to produce cellulase enzyme in natural environments were promising a potential and indigenous strains for bioethanol production in the future [8].
Lipases recently become important enzyme that have chemo, region, and stereo selective properties, which make lipases declared as a one of the most famous enzymes for many industries.Lipases are the third largest group of industrial enzymes that advance used in several industries after proteases and amylases [9,10].Lipases has known widely in nature because lipase can easily produce in yeasts, fungi, bacteria, animal, and plants.Fungal lipases are preferentially applied industrially due to 1255 (2023) 012043 IOP Publishing doi:10.1088/1755-1315/1255/1/012043 2 their high stability and wide substrate specificity [11].Commercial cellulase and lipase were produced from microorganisms that produce a wide variety of extracellular enzymes, mostly from fungi and bacteria are indispensable for the bioconversion of lipids (triacylgylcerols) and cellulose.The enzymes were processed the unique feature of acting at an interface between the aqueous and non-aqueous (i.e.organic) phase; this feature distinguishes them from esterase [12].The Aspergilli are a ubiquitous group of filamentous fungi spanning over 200 million years of evolution.The optimal medium for cellulase and lipase production from Aspergillus are influence the nutrition by the type of substance such as carbon and nitrogen sources, pH condition and growth temperature [13,14].Recent progress in biorefinery and biotechnology have improved the advance technology of enzyme production based on immobilization technique and genetically modification of microorganism and the combination of hybrid process which is combination between biological and chemical process in one step of fermentation [16,17].The Microorganisms exploration as enzyme production has many advantages such as efficient and effective to manage the degradation of raw biomasses and can be used repeatable used in fermentation process [18,19].For industrial process the researchers have continued exploration of new immobilization technique and new enzymes combination for improving enzyme stability for industrial application based on fermentation through the economically feasible aims [20].The future application regarding this research is very important because we can produce enzymes from Indonesian indigenous microbes, and we can use it for supporting local industries and national research.Further research regarding to immobilization process are needed for knowing the optimum condition of combination enzymes that will be use in biofuel industries.

Strain
The source of enzymes cellulase and lipase which is used for industry based on saccharification waste biomass were derivate from fungi and bacteria.Fungi are recommended for those enzymes production because fungal enzymes have extracellular characteristic.This study uses lipolytic fungi Aspergillus niger F538 which is a standard collection of InaCC (Indonesian Culture Collection).Potential fungi Aspergillus niger F538 lare grown on media lipase potato dextrose agar (PDA) and rejuvenated once a weeks.Production of lipase and cellulase from Aspergillus niger indigenous Indonesia as bio-catalyst agent for reducing chemical catalyst demand.The production of each enzyme was using liquid culture with specific substrate such as olive oil and carboxymethyl cellulose furthermore the information of temperature, pH, agitation and production time will be collected for getting the optimum condition of enzyme activities.

Pre-culture and Culture Medium
Preservatives fungi Aspergillus niger F538 with commercial medium PDA contained 39 g/L in aqua distillation solution.Pre-culture and culture medium are contained 39 g/L PDB in aqua distillation, total liquid of 10% olive oil as a fatty source, nitrogen sources such as 0.1 % yeast extract.Pre-culture medium was incubated at 30°C, 200 rpm in agitation for 24 h after inoculated with Aspergillus niger F538 for initiation fermentation process.The composition for producing cellulase is 0.5% carboxymethyl cellulose as a carbon source, 0.075% peptone, 0.05% yeast extract.Culture medium which is inoculated with isolate was incubated at 30°C, agitation 200

Determination of Lipolytic Activity
Lipolytic activity from Aspergillus niger F538 was determined according to the modified method of Krieger et al. (1992).The lipase assay was performed by using 4-nitrophenyl palmitate (4-NPP) as substrate.The substrate solution was prepared by adding solution A (3 mM 4-NPP in 2-Propanol as a solvent) and solution B (0.4 % triton X-100, 0.1 % gum Arabic, 20 ml phosphate buffer 0.02 m pH 7.00 as a solvent) then the final solution of substrate is comparison A:B = 1:9, drop wise with intense stirring.The substrate solution was remained stable for at least 2 h.The assay solution is mixture of 950 μL substrate and 50 μL sample incubated for 20 min at 37°C.The esterase process was de-activated at 95-100 °C for 10 min, next step is centrifuging the mixture at 4 °C, 12,000 rpm for 3 min.Supernatant is p-nitrophenol that released from reaction was measured at 410 nm.The reagent blank contained distilled water instead of enzyme.Standard curve using 4-NPP in 2-propanol with concentration 2, 4, 6, 8, 10, 12 mM) incubated at 37°C for 20 min then measured at 410 nm.One unit of lipase activity is expressed as μmol of ρ-nitriphenol released per minute under the standard assay conditions.

Determination of cellulolytic activity
Cellulase activity were assayed in a reaction mixture containing 0.5 ml of 0.5% CMC in 20 mM phosphate buffer and 0.5 ml of cellulase solution then incubated at room temperature for 30 minutes, continuing with 1.5 ml DNS reagent addition for deactivated the catalytic activity.The catalytic activity was measured the amount of reducing sugar by spectrophotometer at 540 nm.The reducing sugar was determined by Dinitrosalicylic acid (DNS) modification method (Miller et al, 1959) by comparing with glucose as a standard.One unit of the cellulase activity was defined as the amount of enzyme liberating 1 µmol of reducing sugar per minute [15].2.6.Raw Material and spray drying processing.The spray dryer was supplied with concentrated lipase and cellulase at 4 °C.The concentrated lipase and cellulase were mixed with 10% maltodextrin then homogenized at 15,000 rpm for 5 min continues with filtration proses using a 500 µm mesh.The drying process of enzymes were carried out using a spray dryer with an atomizer disk (Vibrasec S A) with a water evaporation capacity of 1.5 L h-1 and it possible to control the inlet and outlet air temperature.The atomizer disk highly speed while the mass feed was kept constant in the process of drying.The drying equipment was operated under vacuum pressures and final product was powder from the cyclone after the process.

Result and Discussion
The characteristic of potential fungi Aspergillus niger InaCC F538 for producing cellulase and lipase for biofuel synthesis.The various enzymes will be used for bioethanol and biodiesel production in advances process for biofuel reaction, due to the simultaneous and continuously reaction in synthesis of biofuel the stabile biocatalyst was needed.Immobilization is the one of solution to improving the stability of enzyme activity.This research obtains the commercial enzymes from local microbe become more stable and can be applied in several industries which is use saccharification and transesterification in their process.

Cellulase Activity
The potential Aspergillus niger InaCC F538 was indigenous microbe from Indonesian culture collection.The isolate can produce cellulase from screening process by using congo red method because it shows clear zone area which is reflect the saccharification process from cellulose became glucose.The production and saccharification process and growth of cell from the microbe was studied by using carboxymethylcellulose as a substrate.The sampling process was obtained for 96 hours incubation time.The principal of determination of cellulase activity is in certain concentration range the amount of reducing sugar of each time incubation and brown color present a positive correlation after the cellulase hydrolysis the cellulose was formed the reducing sugar.

Lipase Activity
Lipase is used to hydrolyze the fats into fatty acid which is needed by microorganism for metabolism.Lipase is constitutive enzyme that constantly expressed without any inducer and lipase expression increases when microorganisms towards the death phase because of the fat amount was become a product of dead cells and obtain increased [5].

Figure 2. Lipase activity from Aspergillus niger InaCC F 538
The characteristic of Aspergillus niger InaCC F 538 to produce lipase is an extracellular enzyme which is collected from fermentation medium.Figure 2 shows the incubation time range 0-96 h, the activity of lipase was increasing simultaneous until reach the optimum incubation time 24 h with value 3.710 U/ml.After 24 h incubation the enzyme activity was decreasing continuously until reach the stabile activity.

Optimization pH and temperature of cellulase
The optimum temperature of crude enzyme and immobilized cellulase were determined by incubating the enzyme in various temperatures (30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C) for 5 min.The temperature stability of each enzyme was determined by incubating the enzyme in the same temperature condition for 1 h and assaying the residual activity using DNS method.The optimum temperature of cellulase activity has shown in Figure 3a and Figure 3b.Both of data were showed the highest value of enzyme activity is in temperature 50 °C with incubation time 24 h and 48 h such as 2.77 U/ml and 3.14 U/ml.The cellulase activity were obtained for 24 h and 48 h incubation time and the result were showed the pH stability of each enzyme was determined at 4 °C optimum condition for 1 h.The highest activity of cellulase was obtained in pH 7 in 48 h incubation time such as 1.72 U/ml.The optimum temperature and pH data can be used for determining glucose production in saccharification process in next bioethanol experiment.

Optimization temperature and pH of Lipase
The optimum pH and temperature were determined by incubating the lipase in optimal range of pH and temperature based on preliminary research and literature because the lipase enable to less active by the variety of several condition such as pH, temperature, ionic strength, denaturation agent, pressure, and mechanical forces.The pH dependence of lipase activity was showed in various pH conditions of 3-10 °C. Figure 4a shown the lipase enzyme from incubation time 24 h and 48 h was studied.The Figure 4a shows the optimum lipase activity in pH range 7 and 8 for 24 h and 48 h incubation time, the value of enzyme activity in pH 7 such as 1.594 U/ml and 1.280 U/ml in pH 8 such as 1.609 U/ml and 1.678 U/ml.The pH 8 showed better activity and more stable in both time incubation.The optimal temperature of lipase activity was tried in various temperatures range of (30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C). Figure 4b shows the lipase activity was optimum in 24 h and 48 h incubation time and the optimum temperature for lipase production from Aspergillus niger InaCC F538 is in 50 °C with value 24 h and 48 h such as 2.742 U/ml and 3.294 U/ml.

Immobilization
The cellulase and lipase immobilized in maltodextrin were showed the increasing enzyme activity (Table 1).The immobilized of both enzymes showed an efficient and effective in preservation also in application for the next saccharification process in biofuel production such as bioethanol and biodiesel.The optimal condition of immobilized enzymes is neutral pH such as pH 7 and temperature 50 °C.The applicable enzyme in industry must have important factors such as stable, optimal in certain temperature and high activity, all the factors will affecting the success of saccharification and fermentation process which was applied in industry applications.The immobilization process enables to enhancing the effectivity and efficiency of enzymes such as preservation and application during the fermentation process.

Conclusion
The immobilization technique using spray drier and filling agent maltodextrin were established in this research for supporting the stability of enzymes product when it was used in biofuel industries.The effectives and efficiency of enzymes product cellulase and lipase in powder can be used continuously in saccharification raw biomass and transesterification process for biofuel industries.Recent progress in biorefinery and biotechnology have improved the advance technology of enzyme production based on immobilization technique and genetically modification of microorganism and the combination of hybrid process which is combination between biological and chemical process in one step of fermentation.The Microorganisms exploration as enzyme production has many advantages such as efficient and effective to manage the degradation of raw biomasses and can be used repeatable used in fermentation process.Several research methods comprise as gene modification microbes, enzymes saccharification, fermentation technology and immobilization enzymes which are important to enhance microbial cellulase and lipase stability and activity.Enzymatic technology to reach possibility of the continuous use or reuse the biocatalyst in production process was needed in modern industry that promotes environmentally friendly technology.

4
The result obtained in this work allowed of showing the cellulolytic activity in Figure1.The data shows that cellulase was optimum in 24 h incubation time with value of activity 4.225 U/ml and simultaneously decrease in 48 h and 72 h such as 3.653 U/mL and 3.331 U/mL.

Table 1 .
Comparation of Immobilization content and yield from cellulase and lipase from potential Aspergillus niger InaCC F538