Preliminary Study of Actinomycetes as Potential Biocatalyst in Biodiesel Production Through Microbial Lipase Activity

Biocatalysts are biological substances that act as an accelerator of chemical reactions without causing influence on the living organism chemically. Biocatalysts based on renewable sources, biodegradable, tolerance to pH and temperature, and high selectivity to stereochemistry substrates and products. The utilization of biocatalyst is environmentally friendly and effective in production costs. This research aimed to determine the potential of actinomycetes as a biocatalyst in biodiesel production. This research method isolation and identification of actinomycetes isolates, conducting lipase activity test to determining lipase enzyme production of actinomycetes isolates. The data analysis in microbial identification was conducted by molecular identification by Gene bank through Basic Local Alignment Search Tool (BLAST). The lipase activity of actinomycetes analyzing by the value of lipase rate of test microbes. Based on chemical analysis of lipase activity, this research results show that actinomycetes T1A has the highest lipase activity by 14.4640 mU/g. Based on molecular identification analysis, actinomycetes T1A was identified as Streptomyces sp., 99% similar to Streptomyces phaeochromogenes.


Introduction
Energy in Indonesia has suffered an energy crisis. For the last years, Indonesia has been imported fossil fuels to fulfill society's needs which no longer fed by national production. The government increases domestic fuel production by exploring and developing various types of national energy resources to achieve Indonesia's energy resilience and independence. One of the efforts is the development of new renewable energy. Based on the geographic condition of Indonesia, which are situated in equator line and as a tropical country, It has an enormous of variable renewable energy potential that spread out across the country, including solar energy sources, water energy and micro-hydro, wind energy sources, geothermal energy sources, ocean wave energy sources, biomass energy sources, and waste to energy (Saefulhak et al., 2017). In sustainable development, exploration and energy consumption of renewable energy has the potential for efficiency and energy conservation fulfilling reasonable and clean energy goals. In addition to that, the exploration, development, and consumption variety of national renewable energy potential will help achieve Indonesia's resilience and independence energy.
Microorganism-producing lipase is studied and has recently become a novel enzyme production focus due to its successful application in widely industrial sectors. A wide range of bacteria, fungi, and other microorganisms produce hundreds of lipase enzymes (Chandra et al., 2020;Andualema & Gessesse, 2012). Actinobacteria or actinomycetes are lipase-producing microbes (Fathurrahman, A.T., 2019). Actinomycetes or actinobacteria are a filamentous gram-positive bacteria phylum, abundantly in most soil, decomposition of organic material, produce a wide range of chemical products successfully used in a variation of industrial application (Prakash et al., 2013;Vaijayanthi et al., 2016). Of over 22.000 reported biobased active substances of microbial metabolites, 45% are derived from actinobacteria, of which threefourths of the total are produced from Streptomyces sp. (Anteneh et al., 2019;Barka et al., 2015). Actinomycetes found in extreme habitats produce novel enzymes with huge commercial industry potential (Mukhtar et al., 2017), Actinobacteria or actinomycetes also in recent robust exploration and development in bioenergy sectors (El-Sheekh et al., 2017;Hama et al., 2018;Sasso et al., 2016;Ayaz et al., 2015;Lan et al., 2016).

Sample Collection
Soil samples were collected from five different succulent plants rhizospheres ( Figure. 1) which are Euphorbia sp. 1, Echinocactus grusonii, Deuterocohnia sp., Euphorbia sp. 2, and Escheveria sp. in about 8 to 10 cm depth, collected into sterile plastic bags and stored in refrigerator at 4°C before isolation process .

2.3.
Actinomycetes Lipase Activity Preliminary Screening 5 out of 10 isolates are randomly selected for lipase activity preliminary screening. The propagation of actinomycetes culture in medium GDP for lipase analysis and actinomycetes selected are grown in 50 mL medium GDP (OXOID CM 129), then incubated in 30oC for 5-7 days.
The determining of Lipase activity was tested by a method based on the increased levels of free fatty acids in the sample (Palilingan et al., 2013). 3 g of CPO and 1 g of polyvinyl alcohol are dissolved in a 40 mL buffer phosphate-citrate pH 5 and supplemented with a 1 mL solution of the enzyme. The sample was incubated at room temperature (25-30°C) for 30 minutes. The reaction is stopped by adding 20 mL of acetone mixture: ethanol (1:1 v/v). Then the sample was titrated using NaOH 1 N using the phenolphthalein indicator until the endpoint was pink. Blanco, the same procedure is carried out without treating enzyme addition (Tri-Panji et al., 2019). Free fatty acid levels are calculated using the following equation: Free fatty acid levels = Lipase activity is calculated using the following equation:

Actinomycetes Identification
Selected actinomycetes isolates were identified by PCR and sequencing method (Gene aid). Identification based on gene encoding 16S ribosomal RNA, which is the most frequently used in bacterial and archaea identification due to slow changes of gene evolution, exist almost in all bacterial genome and contain around 1500bp with various specific functional domain (Michael Janda and Abbott, 2019). DNA sequence fragments obtained were analyzed in Gene bank through the BLAST method, whereas specific similarity regions in the collected sequence were located and compared to find matches in the existing gene database then statistically calculated insignificance (Bazinet et al., 2018;Ladunga, 2017;Wijayanti et al., 2020).

Isolation of Actinomycetes
Succulents are plants commonly lived in extremes conditions, usually drought regions with water scarcity to the nonexistence of water as in arid and semi-arid areas. They developed morphologically and physiologically structure and condition to adapt to extreme habitat conditions for survival. A recent study indicates succulent plants survival and adaptation could derive from plants and microorganisms associations (Nessner Kavamura et al., 2013;Fonseca-García et al., 2016;Karray et al., 2020;De Lyra et al., 2021;Bezerra et al., 2017).
Soil ecosystem has many influential and important components, one of its are soil microorganisms which naturally act as a degrader in various processes of organic material degradation, charge in biotransformation various soil chemical substances and other elements, take the role in nutrients distribution and recycling process essential in animals, plants, other organisms growth, and development . Soil microorganisms exist in various groups microorganisms, commonly found in bacteria, fungi, actinomycetes or actinobacteria, algae, and protozoa groups (Susilawati et al., 2013). Various groups of bacteria, fungi, and actinomycetes or actinobacteria included as an endophytic organism which describes mutualism association of plants and microorganism in the effect of microorganism colonization in root plant it helps plants growth optimally by assisting absorbing essential nutrients from the soil and as plant immune defense by secreted various active secondary metabolites. Contextually, Rhizhosphere describes soil microorganisms and higher plants association in root systems. It concludes of plant root area and soil surface layer of rhizospheres in which high microbiological activity commonly occurs (Baliyarsingh et al., 2017). The soil and vegetation condition greatly influenced rhizosphere microorganism community diversity and its movement .
Actinomycetes are colonized in various types of habitats. In the soil ecosystem, Actinomycetes are one of the rhizosphere microorganism communities and other microorganisms, including bacteria, fungi, algae, and protozoa. The environmental and also physicochemical conditions, including soil type and condition, organic substances, cultivation, and land use, habitat geographically, temperature, pH, salinity, aeration, and moisture soil affected the abundance and variation of actinomycetes existence in any particular soil ecosystem (Arifuzzaman et al., 2010;Zanane et al., 2018).
Actinomycetes were present in all soil samples, the number of actinomycetes isolates abundantly varied in each soil sample. Plate culture dilution ranging from 104-107, based on colony actinomycetes grow on each plate vary in size and appearance and even some culture visualized to have the antimicrobial ability ( Figure. 2). This shows rhizosphere is a natural habitat for actinomycetes. It has rich nutrition for the actinomycete's growth and development. Actinomycetes originated from rhizosphere reported to have antimicrobial compounds (Muleta & Assefa, 2018;Apsari et al., 2019) and potentially as alternative biofertilizer for growth promotion and various agro-active compounds (Anwar et al. 2016) as before explained that actinomycetes found in extreme habitats (as in succulents plant in drought habitat) produce novel enzymes with huge commercial industry potential (Mukhtar et al., 2017) that could be explored for production. 5 colony cultures were isolated to further study in the lipase activity test. The selection of actinomycetes was based on size, Aerial hyphae color, and antimicrobial activity. Those cultures are actinomycetes T1A, T1B, T3D, T3C, and T5A (Figure 3). Based on the visual appearance of colony culture on aerial hyphae color, T1A isolates have a reddish-brown colony appearance, T1B isolated culture has a sly red colony appearance with a bulge in the middle of the colony. T3D remote colony culture has a white colony color with a bump structured in the middle of the settlement. T3C isolated colony culture has an appearance that resembles T3D isolate colony culture, and however single colony has a flat side. Meanwhile, the T5A colony culture has a whitish colony color with a structured, spotted settlement with a distinctive surface of reddish-brown bacteria.

Lipase Activity Test of Actinomycetes
Lipase enzymes are usually derived from many organisms and take part in the metabolism of oils and fats. Lipases take charge of the lipid's metabolism, depositions, and transfers. It also has outstanding catalytic ability and stability in no aqueous media, variety class in region-selectivity, and enantiomerselectivity has successfully and widely applied in many applications of organic synthesis and various reactions, including alcohol lysis, amino lysis, acid lysis, and esterification or transesterification (Gog et al., 2012).
Mainly lipases produced are derived from microbes. Before being selected for lipase production, Lipase microorganisms were isolated and tested for lipolysis ability. Various factors are influential in Lipase production. Several nutrients, mixture ratio, metal ions, pH, agitation, temperature, water content, dissolved oxygen, and timely reaction. The methods used for lipase purification are generally stratified by applying ultrafiltration or ammonium sulfate precipitation to the concentration of culture media, at a later stage with further purification using either one of the chromatography techniques, membrane processes, gel filtration, or immunological techniques of purification. (Luna, C et al., 2016).
Microbial lipase selection In biodiesel conversion becomes an essential step because of lipase's importance in hydrolysis reactions. Lipase is charged on carboxyl ester bonds in triglycerides when producing fatty acid and glycerol. The region-selectivity of lipases is classified based on hydrolyze ester bonds positions. In addition, the substrate specificity of lipases enables them to differentiate various forms of acyl chains. (Luna, C et al., 2016).
This Lipase Activity Test is analyzed as a preliminary study in determining lipase production in actinomycetes. In biodiesel conversion, lipase is a crucial enzyme in breaking down fatty acid contained in biomass into fatty acid Methyl Ester. Based on the lipase rate, mostly actinomycetes have quite high lipase rate on all isolate exclude actinomycetes T3C only 7.6840 mU/ml. Whereas the other reach above 10.000 mU/g. Actinomycetes T1A is the highest lipase activity rate by 14.4640 mU/g ( Table 1). Based on prior culture, actinomycetes T1A shows un-doubly metabolites ability by clearly visible antimicrobial activity (Figure 4). Microbes with high metabolite production tend to have various agroactive compounds such as protease, lignocellulose, lipase, etc. (Anwar et al., 2016). Identification of Actinomycetes Actinomycetes T1A as the highest lipase activity rate will be studied further in the biodiesel production test. Therefore identification is needed. Culture identification of actinomycetes T1A isolates has a reddish-brown colony appearance. Whereas molecular identification by PCR and sequencing method (Gene aid) based on gene encoding 16S ribosomal RNA, DNA sequence fragment obtained were analyzed in Gene bank through BLAST method, resulting in actinomycetes T1A identified as Streptomyces sp. which is 99% similar to Streptomyces phaeochromogenes (Wijayanti et al., 2020). BLAST result shows in Figure 5. In a recent study, Streptomyces sp. globally reported successfully used in biodiesel production using lipase activity assisting in biodiesel conversion. Research by El-Sheekh et al. (2017) co-culture actinomycetes and microalgae in biodiesel production by the batch system resulting in 93% of Streptomyces fatty acids being successfully converted to biodiesel. Research by Cho, 2012, as lipase enzyme derived from Streptomyces sp. CS326 was extracted for biodiesel production, which are new substances than existing lipases. Research by Ugur et al. (2014) conducted the isolation and characterization of lipase enzymes derived from 300 Streptomyces isolates, producing high lipolytic activity in high-temperature reactions and organic solvents through organic solvents transesterification. Recently reported brand new immobilized lipase derives from marine Streptomyces sp., applied in biodiesel production in reaction with methanol at the non-solvent process (Yuan et al., 2016;Wang et al., 2017).
As in Indonesia, Streptomyces sp. is commonly used in producing chemical medicine, antibiotics, anticancer ability, and any other health-related functional (Rahayu, 2011;Chasanah et al., 2012;Krismawati et al., 2015;Nuzulia et al., 2021). Scientific records of Streptomyces sp. utilization in biodiesel production in Indonesia have not yet been seen so far. However, globally has been a success, as explained before. As a comparison in Tabel 2 will be shown biodiesel yield properties of successful utilization of Streptomyces sp. in biodiesel production study with yield properties values based on several American Society for Testing and Materials (ASTM) standards for liquid fuel such as ASTM: D-2638, ASTM 1310, ASTM 240, etc. (El-Sheekh et al., 2017 and National Standard of Indonesia (SNI) 7182:2015 for Biodiesel. Based on these explanations, therefore, Streptomyces sp. has a high potential to be utilized in biodiesel production as a biocatalyst. Especially in Indonesia, where Streptomyces sp. utilization in biodiesel production is rare.

Conclusions
The present study suggests that actinomycetes derived from the rhizosphere of succulent plants are potentially applicable in biodiesel production based on lipase activity. Mainly Streptomyces has successfully utilized in biodiesel production study applying lipase enzyme.