PRODUCTION OF SECONDARY METABOLITES FROM ACTINOMYCETES ASSOCIATED WITH SPONGES AS ANTIBIOTIC CANDIDATES THROUGH THE CO-CULTIVATION APPROACH

• Main Author: Yusriya Hibatullah, Alnadia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/76278
• Institution: Institut Teknologi Bandung

The production of antibacterial compounds can be carried out using two approaches, a single culture (monoculture) or mixed culture (co-culture). The co-culture approach can produce different antibacterial compounds from the monoculture approach. This is due to synergistic and antagonistic interactions that occur between microorganisms. Production of metabolite compounds by co-culture has the advantage of being able to activate cryptic gene clusters and increase the production of bioactive components and product yields through microbial interactions, to trigger the production of metabolites with varied structures. In addition, wider genetic diversity also allows a co-culture approach to carry out more complex metabolic processes. Based on previous studies, two candidates for actinomycetes bacteria that have the potential to produce antibacterial compounds were used, namely Streptomyces harbinensis A1 and Streptomyces aureofaciens A3 isolated from Ranca Buaya Beach, Garut, West Java. The aims of this study: (1) to identify the physiological abilities of S. harbinensis A1 and S. aureofaciens A3 using Biolog EcoPlateTM; (2) optimizing the ratio of S. harbinensis A1 and S. aureofaciens A3 in producing antibacterial compounds; (3) determine the best antibacterial activity of co-culture fermentation in inhibiting the growth of pathogenic bacteria B. subtilis, E. coli, P. aeruginosa and S. aureus; and (4) to identify the antibacterial compound from the ethyl acetate extract which has the best antibacterial activity from the co-cultivation process. This study was initiated by determining the physiological abilities of S. harbinensis A1 and S. aureofaciens A3 using Biolog EcoPlateTM. The production of antibacterial compounds was carried out with three variations of the ratio A1:A3 = 1:1, 1:4 and 4:1 in ISP4A medium for 20 days, temperature 26 ± 2?C, agitation 150 rpm, initial pH 6.97 ± 0.02, inoculum density ~105 CFU/mL and initial inoculum 10% (v/v). Sampling was carried out every two days. Tests performed included total plate count, measurement of glucose and organic acid concentrations using the HPLC method, and total yield of ethyl acetate extracts. The antibacterial activity was tested by the Kirby Bauer method with an extract concentration of 5 mg/mL against four test pathogenic bacteria. The metabolite extract with the best antibacterial activity was identified using GC-MS. The physiological ability test results showed that S. harbinensis A1 and S. aureofaciens A3 had different physiological abilities. S. harbinensis A1 is very good at producing several enzymes that decompose polymers, carbohydrates, and organic acids, while S. aureofaciens A3 is very good at producing enzymes that decompose all substrate groups in Biolog EcoPlateTM, namely polymers, carbohydrates, carbon phosphates, carboxylic acids, amino acids, and amines. Fermentation of S. harbinensis A1: S. aureofaciens A3 with a ratio of 4:1 gave the best results compared to 1:1 and 1:4 because it entered the idiophase or stationary phase on the fourth day of fermentation with the highest amount of yield in ethyl acetate solvent of 0.361 g/ L on the sixth day of fermentation. Antibacterial activity test results from a 4:1 ratio also showed strong antibacterial activity (inhibition zone diameter 10-20 mm) which was classified as narrow spectrum (P. aeruginosa), while a 1:1 ratio produced strong antibacterial compounds with broad spectrum (B. subtilis, P. aeruginosa and S. aureus). Therefore, the GC-MS test was carried out using ethyl acetate extract in a 1:1 ratio which has broad spectrum activity, on days 2, 6, 8 and 18. The results of the analysis showed that compounds derived from phthalates, pyrimidine derivatives, bicyclic diterpenes, flavonoids, and furan derivatives have potential as new antibiotic candidates. The conclusion of this study is that the co-culture approach can produce different antibacterial variations. To produce antibacterial compounds that have a narrow spectrum, fermentation is carried out with a ratio of 4:1, while to obtain a broad spectrum, it is carried out using a ratio of 1:1. In addition, fermentation with a 1:1 ratio also produced five different groups of compounds with potential as new antibiotic candidates.