Karakteristik Bakteri Penambat Nitrogen dari Rizosfer Area Danau Kaolin, Belitung

Abstract

Danau Kaolin merupakan bekas penambangan timah yang terletak di Desa Nibung, Kecamatan Koba, Kabupaten Bangka Tengah. Lahan bekas tambang memiliki sifat fisik maupun kimia yang buruk bagi pertumbuhan tanaman. Kandungan unsur hara yang rendah pada tanah di sekitar Danau Kaolin menyebabkan makhluk hidup, khususnya tanaman sulit tumbuh di sekitar danau, selain itu juga menyebabkan kerusakan struktur tanah, penurunan jumlah mikroba tanah, dan menurunnya bahan organik. Tanaman membutuhkan unsur hara makro yang digunakan untuk pertumbuhan, perkembangan dan produktivitas tanaman. Nitrogen (N2) merupakan salah satu unsur hara makro yang penting bagi tanaman dalam jumlah yang banyak, namun ketersediaannya terbatas bagi tanaman. Nitrogen di atmosfer tersedia cukup melimpah yaitu terkandung sekitar 78%, akan tetapi nitrogen tersebut masih dalam bentuk molekul yang sebagian besar bersifat tidak reaktif, sehingga tanaman tidak mampu menyerapnya secara langsung. Tanaman hanya dapat menyerap nitrogen dalam bentuk ion amonium (NH4+) atau ion nitrat (NO3-). Oleh karena itu, perlu adanya transformasi nitrogen di udara yang mampu mengubah bentuk molekul sehingga dapat diserap oleh tanaman. Strategi yang dapat dilakukan adalah dengan pemanfaatan mikroba seperti bakteri pemfiksasi nitrogen. Bakteri pemfiksasi nitrogen diketahui memiliki beberapa potensi, diantaranya dapat menyuburkan tanah dan menyediakan unsur hara makro serta banyak dimanfaatkan sebagai pupuk hayati. Peneliti terdahulu telah berhasil memperoleh isolat yang teridentifikasi sebagai bakteri pemfiksasi nitrogen yang berasal dari lahan bekas tambang timah di pulau Bangka. Namun belum banyak laporan mengenai bakteri pemfiksasi nitrogen dari lahan bekas tambang Kaolin. Oleh karena itu, penelitian ini bertujuan mengeksplorasi dan menganalisis keragaman bakteri pemfiksasi nitrogen berdasarkan gen 16S rRNA dan gen nifH dari rizosfer area Danau Kaolin, Belitung. Penelitian ini diawali dengan mengisolasi bakteri dari lima sampel tanah rizosfer. Isolat yang diperoleh dihitung kelimpahannya dan dikarakterisasi morfologi secara makroskopis dan mikroskopis. Isolat terpilih dilanjutkan pengujian kadar amonium secara kualitatif dan kuantitatif. Secara kualitatif isolat bakteri ditumbuhkan pada media semi-solid NFB dan dilihat perubahan warna pada media. Secara kuantitatif kadar amonium diukur menggunakan spektrofotometer dan dihitung laju pembentukan amoniumnya. Isolat potensial yang diperoleh dilakukan pengujian hipersensitivitas untuk mengetahui patogenisitasnya. Selanjutnya, isolat potensial dilakukan identifikasi molekuler 16S rRNA dan deteksi gen nifH yang dipilih berdasarkan tingkat konsentrasi amonium yang tinggi. Sebanyak 55 isolat berhasil diisolasi dan dikarakterisasi morfologinya secara makroskopis dan mikroskopis. Berdasarkan pengujian kualitatif, terseleksi sebanyak 13 isolat bakteri fiksasi nitrogen yang ditunjukkan dengan adanya perubahan warna pada media dari hijau menjadi kebiruan yang mengindikasikan bakteri tersebut memiliki aktivitas nitrogenase. Pengukuran secara kuantitatif memperoleh sebanyak 5 isolat potensial (RBA 1.5, RBA 2.3, RBN 4.1, RBN 4.6, 2 dan RBN 5.6). Konsentrasi amonium tertinggi dihasilkan oleh isolat RBN 5.6 sebesar 7,55 µg/mL melalui inkubasi selama delapan hari dengan laju pembentukan amonium yang dihasilkan oleh sel perharinya adalah sebesar 0,38 µg, sedangkan konsentrasi terendah dihasilkan oleh isolat RBA 1.5 sebesar 0,16 µg/mL dengan laju pembentukan amonium yang dihasilkan oleh sel perharinya adalah sebesar 9,25E-05 µg. Identifikasi molekuler gen 16S rRNA dilakukan terhadap 2 isolat potensial RBA 2.3 dan RBN 5.6. Berdasarkan analisis homologi, isolat RBN 5.6 memiliki kemiripan dengan Burkholderia cepacia dan isolat RBA 2.3 memiliki kemiripan dengan Bacillus aquimaris. Analisis secara molekuler menunjukkan bahwa sekuens parsial gen nifH dari isolat RBN 5.6 teridentifikasi sebagai kelompok gen penyandi protein dinitrogenase reduktase.

Kaolin Lake is a former tin mining area located in Nibung Village, Koba District, Central Bangka Regency. Ex-mining land has poor physical and chemical properties for plant. The low nutrient content in the soil around Kaolin Lake causes living things, especially plants, to have difficulty growing around the lake and causes damage to soil structure, a decrease in the number of soil microbes, and a decrease in organic matter. Plants require macronutrients that are used for growth, development, and productivity. Nitrogen (N2) is an important macronutrient for plants in large quantities, but its availability is limited to plants. Nitrogen in the atmosphere is available quite abundantly, at approximately 78%, but the nitrogen is still in the form of molecules, most of which are not reactive, so plants are not able to absorb it directly. Plants can only absorb nitrogen in the form of ammonium (NH4+) or nitrate (NO3-) ions. Therefore, it is necessary to transform nitrogen in the air to change its molecular form so that it can be absorbed by plants. This strategy can be achieved by utilizing microbes such as nitrogen-fixing bacteria. Nitrogen-fixing bacteria are known to have several potential applications, including the ability to fertilize the soil and provide macronutrients, and are widely used as biofertilizers. Previous studies have succeeded in obtaining isolates identified as nitrogen-fixing bacteria originating from the former tin mine land on the island of Bangka. However, there are few reports on nitrogen-fixing bacteria at Kaolin mining sites. Therefore, this study aimed to explore and analyze the diversity of nitrogen-fixing bacteria based on 16S rRNA and nifH genes from the rhizosphere of the Kaolin Lake area, Belitung. This study began by isolating bacteria from five rhizospheric soil samples. The obtained isolates were counted in abundance and characterized macroscopically and microscopically. The selected isolates were then tested qualitatively and quantitatively for ammonium content. Qualitatively, bacterial isolates were grown on semi-solid NFB media and color changes in the media were observed. Quantitatively, the ammonium content was measured using a spectrophotometer, and the rate of ammonium formation was calculated. The potential isolates were subjected to hypersensitivity testing to determine their pathogenicity. Furthermore, potential isolates were subjected to 16S rRNA molecular identification and nifH gene detection, which were selected based on the high ammonium concentration levels. A total of 55 isolates were successfully isolated and characterized macroscopically and microscopically. Based on qualitative testing, 13 isolates of nitrogen-fixing bacteria were selected, as indicated by a color change in the media from green to bluish, indicating that the bacteria have nitrogenase activity. Quantitative measurements revealed five potential isolates (RBA 1.5, RBA 2.3, RBN 4.1, RBN 4.6, and RBN 5.6). The highest ammonium concentration was produced by isolate RBN 5.6 at 7.55 µg/mL through incubation for eight days with the rate of ammonium formation produced by cells per day was 0.38 µg, while the 4 lowest concentration was produced by isolate RBA 1.5 at 0.16 µg/mL with the rate of ammonium formation produced by cells per day was 9.25E-05 µg. Molecular identification of the 16S rRNA gene was performed on two potential isolates, RBA 2.3, and RBN 5.6. Based on homology analysis, isolate RBN 5.6 is similar to Burkholderia cepacia, and isolate RBA 2.3 is similar to Bacillus aquimaris. Molecular analysis showed that the partial sequence of the nifH gene from isolate RBN 5.6 was identified as a group of genes encoding dinitrogenase reductase.