Struktur Komunitas dan Aktivitas Bakteri Nitrifikasi, Denitrifikasi, dan Nitrat Amonifikasi di Tambak Udang Sistem Intensif

Abstract

Udang merupakan salah satu komoditas unggulan perikanan budidaya di Indonesia. Tambak udang sebagai suatu ekosistem memiliki senyawa nitrogen anorganik utama yang dapat terlarut dalam air, yaitu amonia (NH3), nitrat (NO3-), dan nitrit (NO2-). Permasalahan yang sering terjadi dalam pengelolaan tambak udang yaitu penurunan kualitas air dan sedimen yang disebabkan oleh akumulasi nitrogen anorganik. Kandungan nitrogen anorganik yang tinggi di ekosistem tambak menimbulkan dampak negatif. Senyawa amonia (NH3) dan nitrit (NO2-) dapat menyebabkan keseimbangan ekologi mikrob di dalam tambak terganggu. Upaya pelestarian ekosistem tambak terhadap hal tersebut dapat dilakukan melalui pengendalian mikroba beserta aktivitasnya dalam proses transformasi senyawa nitrogen anorganik. Penelitian ini bertujuan mengkaji struktur komunitas bakteri berdasarkan gen 16S rRNA dan gen fungsional melalui pendekatan metagenomik serta mengkaji aktivitas bakteri nitrifikasi, denitrifikasi, dan nitrat amonifikasi dalam mentransformasi senyawa nitrogen anorganik. Sampel air dan sedimen diambil dari kolam tambak udang sistem intensif milik Balai Layanan Usaha Produksi Perikanan Budidaya (BLUPPB). Pengambilan sampel air dan sedimen dilakukan pada 2 jenis kolam yang berbeda perlakuan yaitu kolam dengan probiotik terpilih dan kolam dengan probiotik komersial (QuickPro dan Boster Biolacto). Pengambilan sampel air dilakukan pada saat umur pemeliharaan udang 0 hari, 15 hari, 30 hari, 45 hari, 60 hari, 75 hari, dan 90 hari. Di sisi lain, pengambilan sampel sedimen juga dilakukan pada saat umur pemeliharaan udang 0 hari, 30 hari, 60 hari, dan 90 hari. Parameter kualitas air dan sedimen meliputi senyawa nitrogen anorganik dianalisis sesuai prosedur APHA. Analisis kelimpahan bakteri nitrifikasi, denitrifikasi, dan nitrat amonifikasi dilakukan menggunakan metode MPN. Isolasi bakteri dilakukan dari medium uji MPN yang positif. Isolat bakteri murni yang diperoleh dikarakterisasi morfologi koloni dan morfologi sel dengan pewarnaan Gram. Isolat murni diuji aktivitas oksidasi amonia dan reduksi nitrat. Isolat dengan aktivitas oksidasi amonia dan reduksi nitrat tertinggi dipilih untuk dianalisis kinetika aktivitas, diamati pola pertumbuhannya, dan diidentifikasi secara molekuler berdasarkan gen 16S rRNA. Di sisi lain, pendekatan NGS dilakukan untuk mengestimasi struktur komunitas mikroba berdasarkan gen 16S rRNA dan gen fungsional. Analisis kelimpahan gen 16S rRNA dan gen fungsional bakteri nitrifikasi, denitrifikasi, dan nitrat amonifikasi juga dilakukan dengan metode qPCR. Hasil analisis kelimpahan gen bakteri 16S rRNA dengan qPCR menunjukkan bahwa kelimpahan gen bakteri 16S rRNA dan nrfA untuk bakteri nitrat amonifikasi pada kolam probiotik terpilih signifikan lebih tinggi daripada kolam probiotik komersial selama masa pemeliharaan udang. Kelimpahan bakteri gen 16S rRNA pada kolam probiotik terpilih berkisar dari 2,11 ± 0,14 × 108 hingga 2,70 ± 0.38 × 108 salinan g sedimen-1, sedangkan pada kolam probiotik komersial dari 1,38 ± 0.26 × 108 hingga 1,87 ± 0.19 × 108 salinan g sedimen-1. Kelimpahan gen bakteri nitrat amonifikasi (nrfA) pada kolam probiotik terpilih berkisar antara 1,87 ± 0.17 × 105 hingga 2,51 ± 0.15 × 105 salinan gen sedimen-1, sedangkan pada kolam probiotik komersial dari 1,63 ± 0.14 × 105 hingga 2,12 ± 0.21 × 105 salinan gen sedimen-1. Struktur komunitas bakteri berdasarkan gen 16S rRNA pada sedimen tambak menunjukkan bahwa Proteobacteria merupakan filum yang paling melimpah di semua sampel dan menyumbang hingga persentase 33-43%, diikuti oleh Bacteroidetes (8,6-17,5%), Firmicutes (6,1-12,6%), dan Chloroflexi (6,1-12,1%). Secara umum, kelimpahan relatif dari filum Proteobacteria dan Bacteroidetes di kolam yang diberi probiotik terpilih lebih tinggi daripada kolam dengan probiotik komersial. Kelimpahan relatif filum Firmicutes di kolam probiotik terpilih lebih rendah daripada kolam dengan probiotik komersial. Hasil analisis pengelompokkan dengan metode UPGMA menunjukkan bahwa komunitas bakteri pada kolam dengan probiotik komersial hari ke-60 (KNP_60) memiliki kemiripan yang tinggi dengan komunitas bakteri pada kolam probiotik terpilih hari ke-60 (KP_60). Struktur komunitas bakteri nitrat amonifikasi berdasarkan gen nrfA pada sedimen tambak menunjukkan bahwa Verrucomicrobia merupakan filum yang paling melimpah di semua sampel dan menyumbang hingga persentase 39,4-64,7%, diikuti oleh Proteobacteria (33,6-53,6%,), dan Terrabacteria (1,5-11,1%). Hasil analisis pengelompokkan dengan metode UPGMA menunjukkan bahwa Komunitas bakteri nitrat amonifikasi pada kolam dengan probiotik komersial (KNP_30) memiliki kemiripan yang tinggi dengan komunitas bakteri pada kolam probiotik terpilih (KP_30). Analisis CCA yang digunakan untuk menentukan hubungan parameter lingkungan dengan komunitas bakteri menunjukkan bahwa amonium dan nitrat berpengaruh signifikan terhadap struktur komunitas bakteri yang ada pada sedimen tambak di kedua kolam probiotik. Berdasarkan pendekatan culturable, sebanyak 29 isolat terseleksi berhasil diperoleh dari air dan sedimen kolam tambak udang sistem intensif, 13 isolat merupakan kelompok bakteri nitrifikasi, 8 isolat termasuk kelompok bakteri denitrifikasi, dan 8 isolat termasuk kelompok bakteri nitrat amonifikasi. Isolat yang memiliki aktivitas oksidasi amonia dan reduksi nitrat terbaik yaitu SwNi27, SwNi44, SwDe15, SwDe22, SWR5, dan SWR8. Kecepatan maksimum (Vmax) oksidasi amonia isolat SwNi27 dan SwNi44 masing-masing adalah 0,09 mM jam-1 dan 0,17 mM jam-1. Nilai Km isolat SwNi27 dan SwNi44 yaitu 0,21 mM dan 0,07 mM. Kecepatan maksimum (Vmax) reduksi nitrat isolat SwDe15, SwDe22, SWR5, dan SWR8 masing-masing adalah 0,11 mM jam-1, 0,09 mM jam-1, 0,23 mM jam-1, dan 0,14 mM jam-1. Nilai Km masing-masing isolat tersebut yaitu 0,41 mM, 0,44 mM, 0,16 mM, dan 0,38 mM. Hasil identifikasi 16S-rRNA isolat SwNi27 memiliki kemiripan sebesar 100% dengan Acinetobacter venetianus, isolat SwNi44 memiliki kemiripan sebesar 99% dengan Acinetobacter sp., isolat SwDe15 dan SwDe22 memiliki kemiripan sebesar 100% dengan Microbacterium sp., isolat SWR5 dan SWR8 memiliki kemiripan sebesar 99% dengan Bacillus vireti.

Shrimp is one of the leading aquaculture commodities in Indonesia. Shrimp ponds as an ecosystem have the main inorganic nitrogen compounds that can be dissolved in water, namely ammonia (NH3), nitrate (NO3-), and nitrite (NO2-). The problem that often occurs in the management of shrimp ponds is the decrease in water and sediment quality caused by the accumulation of inorganic nitrogen. High inorganic nitrogen content in the pond ecosystem has a negative impact. Ammonia (NH3) and nitrite (NO2-) compounds can disrupt the ecological balance of microbes in ponds. Efforts to preserve the pond ecosystem regarding this can be done through controlling microbes and their activities in the process of transforming inorganic nitrogen compounds. This research was conducted to study the bacterial community structure based on the 16S rRNA gene and functional genes through a metagenomic approach, as well as to study the activity of nitrifying, denitrifying, and nitrate-ammonifying bacteria in transforming inorganic nitrogen compounds. Water and sediment samples were taken from the intensive system shrimp pond owned by the Aquaculture Production Business Service Center (BLUPPPB). Sampling of water and sediment was carried out in 2 types of ponds with different treatments, namely ponds with selected probiotics and ponds with commercial probiotics (QuickPro and Biolacto Booster). Water samples were taken at the age of shrimp rearing 0 days, 15 days, 30 days, 45 days, 60 days, 75 days and 90 days. On the other hand, sediment sampling was also carried out at the age of shrimp rearing 0 days, 30 days, 60 days and 90 days. Water and sediment quality parameters including inorganic nitrogen compounds were analyzed according to the APHA procedure. Analysis of the abundance of nitrifying, denitrifying, and nitrate-ammonifying bacteria was carried out using the MPN method. Bacterial isolation was carried out from positive MPN test medium. The pure bacterial isolates obtained were characterized by Gram staining of colony morphology and cell morphology. Pure isolates were tested for ammonia oxidation and nitrate reduction activities. Isolates with the highest ammonia oxidation and nitrate reduction activities were selected to be analyzed for activity kinetics, observed growth patterns, and identified molecularly based on the 16S rRNA gene. On the other hand, the NGS approach was used to estimate the microbial community structure based on 16S rRNA genes and functional genes. Analysis of 16S rRNA gene abundance and functional genes of nitrifying, denitrifying, and nitrate-ammonifying bacteria was also performed by qPCR method. The results of the analysis of bacterial 16S rRNA gene abundance using qPCR showed that the bacterial 16S rRNA and nrfA gene abundance for nitrate-ammonifying bacteria in selected probiotic ponds was significantly higher than commercial probiotic ponds during the shrimp rearing period. The abundance of bacterial 16S rRNA gene in selected probiotic ponds ranged from 2,11 ± 0,14 × 108 to 2,70 ± 0.38 × 108 gene copies of g sediment-1, whereas in commercial probiotic ponds from 1,38 ± 0.26 × 108 to 1,87 ± 0.19 × 108 gene copies of g sediment-1. Gene abundance of ammonified nitrate bacteria (nrfA) in selected probiotic ponds ranged from 1,87 ± 0.17 × 105 to 2,51 ± 0.15 × 105 gene copies of g sediment-1, while in commercial probiotic ponds it was from 1,63 ± 0.14 × 105 to 2,12 ± 0.21 × 105 gene copies of g sediment-1. Bacterial community structure based on the 16S rRNA gene in pond sediments shows that Proteobacteria is the most abundant phylum in all samples and contributes up to 33-43%, followed by Bacteroidetes (8,6-17,5%), Firmicutes (6,1- 12,6%), and Chloroflexi (6,1-12,1%). In general, the relative abundance of Proteobacteria and Bacteroidetes phyla in ponds fed with selected probiotics was higher than ponds with commercial probiotics. The relative abundance of Firmicutes phylum in selected probiotic ponds was lower than ponds with commercial probiotics. The results of grouping analysis using the UPGMA method showed that the bacterial community in ponds with commercial probiotics on the 60th day (KNP_60) had a high similarity with the bacterial communities in the selected probiotic ponds on the 60th day (KP_60). The community structure of ammonified nitrate bacteria based on the nrfA gene in pond sediments showed that Verrucomicrobia was the most abundant phylum in all samples and contributed up to a percentage of 39,4-64,7%, followed by Proteobacteria (33,6-53,6%), and Terrabacteria (1,5-11,1%). The results of grouping analysis using the UPGMA method showed that the community of ammonified nitrate bacteria in ponds with commercial probiotics (KNP_30) had a high similarity to the bacterial community in selected probiotic ponds (KP_30). The CCA analysis used to determine the relationship between environmental parameters and the bacterial community showed that ammonium and nitrate had a significant effect on the structure of the bacterial community present in pond sediments in both probiotic ponds. Based on the culturable approach, 29 selected isolates were obtained from water and sediment of intensive system shrimp ponds, 13 isolates belonged to the group of nitrifying bacteria, 8 isolates belonged to the group of denitrifying bacteria, and 8 isolates belonged to the group of ammonifying nitrate bacteria. The isolates with the best ammonia oxidation and nitrate reduction activities were SwNi27, SwNi44, SwDe15, SwDe22, SWR5, and SWR8. The maximum speed (Vmax) of ammonia oxidation from isolates SwNi27 and SwNi44 were 0,09 mM hour-1 and 0,17 mM hour-1. The Km values of isolates SwNi27 and SwNi44 were 0,21 mM and 0,07 mM. The maximum speed (Vmax) of nitrate reduction isolates from SwDe15, SwDe22, SWR5, and SWR8 were 0,11 mM hour-1, 0,09 mM hour-1, 0,23 mM hour-1, and 0,14 mM hour-1. The Km values of each isolate were 0,41 mM, 0,44 mM, 0,16 mM and 0,38 mM. The results of the identification of the 16S-rRNA isolate SwNi27 had a 100% similarity to Acinetobacter venetianus, isolate SwNi44 had a similarity of 99% to Acinetobacter sp., isolates SwDe15 and SwDe22 had a similarity of 100% to Microbacterium sp., isolates SWR5 and SWR8 were 99 % closely related to Bacillus vireti.