Keragaman Bakteri Indigenous dari Sedimen Mangrove di Perairan Teluk Ambon Maluku

Abstract

Kawasan mangrove di Teluk Ambon tersebar di beberapa wilayah, termasuk Desa Waiheru dan Poka. Sebaran jenis mangrove di Waiheru masih cukup beragam dan mengikuti zonasi alami seperti yang umumnya ditemukan di ekosistem mangrove. Namun, kondisi mangrove di Desa Poka memprihatinkan karena pencemaran akibat limbah antropogenik yang menyebabkan kematian mangrove di daerah tersebut. Penelitian mengenai keragaman bakteri di kawasan ini sebelumnya telah dilakukan menggunakan metode pengkulturan konvensional, di mana bakteri dikultivasi pada medium buatan untuk menghitung koloni dan mengamati morfologi mereka di bawah mikroskop. Namun, hanya sebagian kecil bakteri, yaitu sekitar 0,1-1%, yang mampu tumbuh dalam media kultur, sementara sebagian besar bakteri berada dalam kondisi anabiosis yang membuat mereka sulit dikultur. Oleh karena itu, analisis metagenomik dengan teknologi sekuensing generasi ketiga, seperti Oxford Nanopore Technology (ONT), dinilai lebih efektif dalam mendeteksi seluruh komunitas bakteri, termasuk yang tidak dapat dikultur dengan metode konvensional. Mengatasi keterbatasan metode kultur, penelitian ini bertujuan untuk mengeksplorasi keragaman bakteri indigenous di sedimen mangrove Teluk Ambon, khususnya di kawasan alami dan tercemar. Sampel sedimen diambil dari kedua kawasan untuk dilakukan analisis fisikokimia dan keragaman bakteri menggunakan metode kultur dan non-kultur. Analisis fisikokimia mencakup pengukuran parameter seperti suhu, pH, salinitas, dissolved oxygen (DO), biological oxygen demand (BOD), nitrat, nitrit, sulfat, dan zat organik. Bakteri yang berhasil dikultur kemudian dianalisis morfologinya, sedangkan hubungan antara jumlah koloni bakteri dengan data fisikokimia dianalisis menggunakan Canonical Correlation Analysis (CCA). Isolat yang dipilih berdasarkan INP tertinggi diidentifikasi secara molekuler melalui analisis gen 16S rRNA. Sementara itu, keragaman bakteri unculturable dianalisis menggunakan Next Generation Sequencing (NGS) dengan platform nanopore sequencing. Dari analisis fisikokimia, sedimen mangrove di kawasan alami (Desa Waiheru) dan tercemar (Desa Poka) umumnya memenuhi standar kualitas lingkungan berdasarkan PP No 22 Tahun 2021 untuk biota laut, kecuali nilai BOD di kawasan tercemar. Tidak ada perbedaan signifikan dalam suhu, pH, DO, BOD, nitrat, nitrit, sulfat, dan zat organik antara kedua kawasan, kecuali salinitas yang menunjukkan perbedaan nyata. Metode kultur mengungkapkan bahwa jumlah total bakteri di kawasan alami lebih tinggi dibandingkan kawasan tercemar, dengan masing-masing mencapai rata-rata 6,2 x 104 CFU/g dan 5,5 x 104 CFU/g. Dari total 33 isolat yang ditemukan, 17 berasal dari kawasan alami dan 16 dari kawasan tercemar. Isolat KAT2 dan KTT23 memiliki INP tertinggi di kawasan alami dan tercemar, dengan persentase kemiripan masing-masing 100% terhadap Acinetobacter haemolyticus dan Exiguobacterium acetylicum. Penggunaan metode non-kultur dengan teknologi nanopore sequencing berhasil mengidentifikasi jumlah spesies yang lebih banyak, yaitu 6.837 spesies di kawasan alami dan 6.117 spesies di kawasan tercemar. Sulfurovum aggregans menjadi spesies dominan di kedua kawasan, dengan proporsi sebesar 3,91% di kawasan alami dan 4,56% di kawasan tercemar. Analisis diversitas alfa menunjukkan bahwa diversitas komunitas bakteri lebih tinggi di kawasan alami dibandingkan kawasan tercemar, dengan jumlah spesies yang teramati mencapai 12.954. Sebanyak 28,23% spesies di kawasan alami dan 19,78% di kawasan tercemar merupakan spesies unik, sementara 51,99% spesies ditemukan di kedua kawasan, menunjukkan adanya spesies bakteri yang mampu beradaptasi dengan berbagai kondisi lingkungan. Kesimpulan dari penelitian ini menunjukkan bahwa metode unculturable, yang menggunakan teknologi sekuensing generasi ketiga, mampu mengungkap keragaman bakteri yang lebih komprehensif dibandingkan metode kultur. Meskipun secara umum parameter fisikokimia di kedua kawasan tidak menunjukkan perbedaan signifikan, faktor-faktor ini tetap memainkan peran penting dalam memengaruhi distribusi dan kelimpahan bakteri di sedimen mangrove Teluk Ambon.

Mangrove areas in Teluk Ambon are spread across several regions, including villages in Waiheru and Poka. The diversity of mangrove species in Waiheru remains relatively high and follows the natural zonation typically observed in mangrove ecosystems. However, the condition of mangroves in Poka Village is alarming because of anthropogenic pollution, which has led to the death of many mangroves in the area. Previous studies on bacterial diversity in these regions have been conducted using conventional culturing methods, in which bacteria are cultivated on artificial media to count colonies and observe their morphology under a microscope. However, only a small fraction of bacteria, approximately 0.1-1%, can grow on culture media, while most bacteria remain in a state of anabiosis, making them difficult to culture. As a result, metagenomic analysis with third-generation sequencing technology, such as Oxford Nanopore Technology (ONT), is considered more effective in detecting the entire bacterial community, including those that cannot be cultured using conventional methods. To address the limitations of culturing methods, this study aimed to explore the diversity of indigenous bacteria in the mangrove sediments of Teluk Ambon, specifically in natural and polluted areas. Sediment samples were collected from both areas for physicochemical and bacterial diversity analyses using culture and non-culture methods. Physicochemical analysis includes measuring parameters such as temperature, pH, salinity, dissolved oxygen (DO), biological oxygen demand (BOD), nitrate, nitrite, sulfate, and organic matter. The cultured bacteria were then morphologically analyzed, and the relationship between the number of bacterial colonies and physicochemical data was analyzed using Canonical Correlation Analysis (CCA). Isolates selected based on their highest INP were molecularly identified using 16S rRNA gene analysis. Meanwhile, the diversity of unculturable bacteria was analyzed using next-generation sequencing (NGS) with the nanopore sequencing platform. Physicochemical analysis revealed that mangrove sediments in the natural (Waiheru Village) and polluted (Poka Village) areas generally met environmental quality standards based on Government Regulation No. 22 of 2021 for marine biota, except for the BOD value in the polluted area. There were no significant differences in temperature, pH, DO, BOD, nitrate, nitrite, sulfate, and organic matter between the two areas, except for salinity, which showed a notable difference. The culture method revealed that the total number of bacteria in the natural area was higher than in the polluted area, with averages of 6.2 × 104 CFU/g and 5.5 × 104 CFU/g, respectively. Of the 33 isolates found, 17 were from natural areas and 16 were from polluted areas. Isolates KAT2 and KTT23 had the highest INP in natural and polluted areas, with 100% similarity to Acinetobacter haemolyticus and Exiguobacterium acetylicum, respectively. Using the non-culture method with nanopore sequencing technology, a greater number of species were identified, with 6,837 species in the natural area and 6,117 species in the polluted area. Sulfurovum aggregans was the dominant species in both areas, accounting for 3.91% in the natural area and 4.56% in the polluted area. Alpha diversity analysis indicated that bacterial community diversity was higher in the natural area than in the polluted area, with the observed species count reaching 12,954. A total of 28.23% of species in the natural area and 19.78% in the polluted area were unique species, whereas 51.99% of species were found in both areas, indicating the presence of bacterial species capable of adapting to varying environmental conditions. The conclusion of this study shows that the unculturable method using third-generation sequencing technology can reveal a more comprehensive bacterial diversity than the culture method. Although the physicochemical parameters in both areas generally did not show significant differences, these factors still played an essential role in influencing the distribution and abundance of bacteria in the mangrove sediments of Teluk Ambon.