ISOLATION AND IDENTIFICATION OF BACTERIA REMOVING NITRITE, NITRATE, AND AMMONIUM FROM BIOBALLS FILTER

The presence of effective bacteria removing nitrite, nitrate, and ammonia in a recirculating aquaculture system (RAS) is necessary to attenuate their toxicity to fish. The research was conducted to find bacteria that can be cultured and reduce nitrite, nitrate, and ammonium. Sixteen bacterial colonies were isolated from bioballs of RAS biofilter and tested for their ability to reduce nitrite or nitrate concentrations. Using a simple indicator paper for nitrite and nitrate, four isolates that reduced nitrite and nitrate concentrations, namely K1NA3, K2NA3, CNA1, and PRO4NA1 were selected. The four isolates were then evaluated for the metabolism of nitrate, nitrite, and ammonium compounds using the spectrophotometry method. Results showed that the isolates K1NA3, CNA1, and PRO4NA1 reduced nitrite concentration but produced ammonium, whereas K1NA3 isolate was able to reduce nitrate concentration but produced both nitrite and ammonium. Experiments in reducing ammonium levels in the synthetic waste media showed the ability of four isolates to reduce ammonium levels after six days despite producing nitrite. Based on the 16S rRNA gene analysis, these isolates have a close relationship to Pseudomonas otitidis (K1NA3 and K2NA3), Acinetobacter cumulans (CNA1), and Vogesella perlucida (PRO4NA1).


INTRODUCTION
Excessive nitrogen-based waste in the form of ammonia, ammonium, nitrite, and nitrate can be harmful to ecosystems, both marine and freshwater, human, and animal health (Kumar et al., 2020). The accumulation of inorganic nitrogen is the main obstacle in an intensive aquaculture (Khangembam et al., 2017).
In aquaculture, bacteria play a role in the transformation of nitrogen compounds (Hu et al., 2013). This group of bacteria can convert N-organic compounds into ammonia (NH 3 ). Ammonia is then oxidized to nitrite (NO 2 ) and nitrate (NO 3 ) in the nitrification process (Stein & Klotz, 2016). The nitrate formed can be reduced to nitrogen gas (N 2 ) through denitrification or converted back to ammonia. The formation of nitrate into nitrogen gas is the ideal transformation in aquaculture systems (Rassamee et al., 2011). The utilization of indigenous microbes in the pond is considered an environmentally friendly method to accelerate nitrogen waste removal. The more active bacterial inoculum is usually in the form of a consortium (Barik et al., 2018). Each strain of bacteria has different metabolic capability in metabolizing nitrogen, mineralizing organic matter, and removing toxic compounds. (Dahiya & Mohan, 2016).
The development of wastewater treatment from recirculating aquaculture systems (RAS) to treat residual harmful nitrogenous waste compounds from RAS, in which bacteria play a role in nitrification and simultaneous nitrification-denitrification, is still needed (Shitu et al., 2021). Isolation of bacteria that reduce ammonia, nitrite, and nitrate levels is still considered relevant to be carried out in the context of controlling nitrogen waste on an industrial scale (Motamedi & Jafari, 2020). Screening of such bacteria is expected to increase the effectiveness of using indigenous bacteria to reduce nitrogen compound Isolation and identification of bacteria removing nitrite ..... (Qoriatul Ilma) waste. This study aims to obtain bacterial colonies that have the potential to reduce the content of ammonium, nitrite, and nitrate.

MATERIALS AND METHODS
Briefly, the process of obtaining effective bacterial isolates begins with the isolation of bacteria from the bioballs filter. The isolates were then grown on 4 mL of NB media for 24 hours and centrifuged. The pellets obtained were added with 200 µL nitrite (NaNO 2 ) 5 mg/L or nitrate (KNO 3 ) 20 mg/L, and incubated for 24 hours. The decrease in nitrite or nitrate content in the supernatant indicated the capability of the isolates in removing nitrite or nitrate respectively and was estimated using indicator paper. Bacterial colonies capable of reducing nitrite and/or nitrate content were then selected for further analyses.
The supernatant containing nitrite or nitrate in which isolate is considered capable of reducing nitrite and/or nitrate was then measured using the spectrophotometer method to confirm the ability of the bacteria to reduce the levels of nitrite and nitrate. Ammonium production, as well as nitrate production in nitrite supernatant and nitrite production in nitrate supernatant, were also measured. The isolates capable of reducing nitrite or nitrate with low ammonium production were then selected for the ammonium reduction test on synthetic waste media by growing single or mixed isolates in the media containing ammonium based on a modified method of Du et al. (2015).

Isolation of Bacteria
Bacteria were isolated from bioballs used as a biological filter in a recirculating aquaculture system at the Research Center for Biology, National Research and Innovation Agency (BRIN), Cibinong, West Java. Samples from bioballs were taken at approximately 0.1 g by scraping the bioball cavity using a sterile spatula. The bacteria were grown on nutrient agar (NA) and nutrient broth (NB) media (Himedia) at room temperature.

Qualitative Measurement of Bacteria Activity to Reduce Nitrite and Nitrate Using Indicator Paper (Screening)
Bacterial isolates were grown in 4 mL of NB media for 24 hours with agitation at 115 rpm at room temperature. Cell suspension of 1 mL was centrifuged for five minutes at 10,000 rpm. The pellets were washed and added 200 µL of 5 mg/L NaNO 2 solution for the nitrite reduction test or 20 mg/L KNO 3 for the nitrate level test. Microtubes were agitated for 24 hours. The sample of 3 µL was placed on nitrite-nitrate test paper (JBL EasyTest, Germany) for 3-5 minutes to detect any color change on the test paper. Bacteria whose inoculum was positive for reducing nitrite or nitrate that showed pink to white color were selected for further testings.

Determination of Nitrite and Nitrate Metabolism
The ability of single isolates to decrease nitrite or nitrate in media, production of ammonium, and production of nitrate or nitrite in media containing nitrite or nitrate were observed to confirm the occurrence of nitrite and nitrate metabolic processes. Isolate cultures of 24 hours were centrifuged at 5,000 g; the pellet was washed with distilled water. The pellets were then resuspended with 2 mL of nitrite or nitrate with OD 600 nm of 3.75 and 2.26, respectively. Cultures were performed in microplate 12 wells, incubated at room temperature with shaking at 100 rpm. After six days of incubation, the nitrite, nitrate, and ammonium levels were measured using the spectrophotometer method. Experiments were performed in triplicate.

Measurement of Nitrite Concentration (SNI 06-6989.9, 2004)
Measurement of nitrite concentration was performed using the azo staining method. A sample of 10 µL was added to 240 µL of distilled water into a 96-well microplate. The diluted sample was added with 5 µL of sulfanilamide acid and 5 µL of NED solutions and then homogenized. The mixture was allowed to stand for 10 minutes and then measured using a Indonesian Aquaculture Journal, 17 (1), 2022, 13-22 microplate reader with a wavelength of 543 nm. The calibration curve was prepared by preparing a solution of NaNO 2 as a standard solution of nitrite with a concentration from 0 to 0.25 mg/L.

Measurement of Nitrate Concentration (EPA Method: 352.1, 1971)
The measurement of nitrate was carried out by the brucine sulfate colorimetric method. A sample of 40 µL was added with 360 µL of distilled water. The diluted sample was added with 80 µL of 30% NaCl solution, 400 µL of 75% sulfuric acid, and 20 µL of brucine sulfate reagent. The mixture was homogenized and heated at 95°C for 20 minutes. Then the absorbance was measured at a wavelength of 410 nm. The calibration curve was made by preparing a solution of KNO 3 as a standard solution of nitrate concentration from 0 to 2.5 mg/L.

Measurement of Ammonium Concentration (EPA Method: 350.2, 1974)
Measurement of ammonium concentration was done by using the Nessler method. The sample of 25 µL was added with 225 µL of distilled water in a 96well microplate. The diluted sample was added with 2.5 µL Nessler A and 2.5 µL Nessler B, homogenized and allowed to stand for 10 minutes. The absorbance of the mixture was measured at a wavelength of 490 nm. The calibration curve was carried out by measuring the OD of a standard solution of ammonium with a concentration from 0 to 5 mg/L.

Identification of Bacteria
Selected bacteria were identified molecularly based on the partial 16S rRNA gene. The colony PCR technique was carried out by preparing the mixture (total 30 uL) consisting of 11 uL ddH2O (nuclease-free water), 15 uL Go Taq Green Master Mix 2x, 2 uL 10 pM forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and 2 uL 10 pM reverse primer 1492R (5'-CGG TTACCTTGTTACGAC TT-3'). One bacterial colony grown on nutrient agar was picked up and loaded into the PCR mixture in the microtube. The PCR condition included the following steps: initiation at 94°C for five min; the 30 cycles of denaturing at 94°C for one min, annealing at 55°C for one min, and extension at 72°C for one min; and the final elongation at 72°C for one min. The 16S rRNA sequences of each bacterial isolate were compared with homologous sequences using the BLAST (Basic Local Alignment Search Tool: http://blast.ncbi.nlm.nih.gov/). A phylogenetic tree was constructed using Clustal X based on the neighbor-joining method.

Isolate of Bacteria and Its Ability to Reduce Nitrate and Nitrite Content
There were 16 bacterial colonies visually observed and differed in morphology. The ability of each colony to reduce nitrate and nitrite content is presented in Figure 1 and Table 1. The white color of the band in Figure 1 indicated that the content of nitrate (band at the top) or nitrite (band at the bottom) decreased. From the data, it can be seen that there are four potential isolates, namely CNA1, K1NA3, K2NA3, and PRO4NA1. The ability to reduce nitrite and nitrate content was then confirmed using the spectrophotometer method.
In order to overcome poisoning due to nitrogenbased compounds, efforts are made to reduce the levels of ammonium, nitrite, and nitrate; which are then expected to become nitrogen molecules (N 2 ). Since the chemical reaction in the process of transformation of ammonium to nitrite, then to nitrate is reversible (Blum et al., 2018), it is necessary to know the potential of bacterial isolates in converting nitrite to nitrate and vice versa and converting ammonium to nitrite or nitrate and vice versa.

Nitrite Metabolism
It was confirmed that three isolates were able to decrease nitrite, namely K1NA3, CNA1, and PRO4NA1 ( Figure 2). However, one of the products of nitrite metabolism is ammonium (Figure 3). In nitrite solution, K1NA3 isolate produced the highest ammonium. However, nitrite metabolism in these isolates did not produce nitrate (nitrate content was not observed, data not shown).

Nitrate Metabolism
Two isolates were confirmed to be able to reduce nitrate concentration, namely K1NA3 and PRO4NA1 (Figure 4). PRO4NA1 isolate was the highest in reducing nitrate but produced a higher concentration of nitrite than K1NA3 ( Figure 5). In addition to producing nitrite, nitrate metabolism can produce ammonium, especially in K1NA3 isolate ( Figure 6).

Ammonium Metabolism by Bacterial Consortia
Ammonium is an inorganic nitrogen compound resulting from the breakdown of protein. Considering that bacteria in an ecosystem contain nitrogenous waste that plays a role in the nitrogen cycle, which is usually complex, the ability of the bacterial consortium to remove ammonium and its metabolic pro-Isolation and identification of bacteria removing nitrite ..... (Qoriatul Ilma) ducts is important to anticipate the accumulation of nitrogen compounds that can harm organisms such as nitrite, nitrate or even N 2 O in the greenhouse effect (Blum et al., 2018). Figure 7, showed that isolates of PRO4NA1, B bacterial mixture (CNA1 and PRO4NA1) and the mixture of the four isolates could reduce ammonium levels in six days. These data illustrated that the application of these bacterial isolates to nitrogen waste of freshwater aquaculture could reduce ammonium contained within. However, the reduction was followed by the accumulation of nitrite (Figure 8), indicating the ineffectiveness of denitrification. The bacterial mixture of four isolates demonstrated the highest accumulation of nitrite. How-ever, until day-6, no nitrate was observed (data not shown).

Identification of Bacterial Isolates
PCR amplification of four isolates (K1NA3, K2NA3, CNA1, and PRO4NA1) was successfully carried out based on observations on gel electrophoresis at around 1,500 bp. Partial sequencing of the 16S rRNA gene resulted in a sequence length ranging from 1,005-1,392 bp. Nevertheless, it provided an overview of genetic similarity information through alignment with a collection of rRNA sequences from NCBI Gen bank. BLAST analysis showed the percentage similarity of all isolates was 98.20%-99.75% (Table 2).     The phylogenetic analysis of 1,000 bootstraps showed that four isolates were grouped into three different clades (Figure 9), namely Pseudomonas, Acinetobacter, and Vogesella.
P. otitidis was first isolated and identified from the clinical specimens of infected human ears. The cells are rod-shaped, motile, and Gram-negative (Clark et al., 2006). According to Dahiya & Mohan (2016), P. otitidis is a potential bacterium for wastewater treatment. (Dados et al., 2015;Wu et al., 2009). Pseudomonas is a member of Gammaproteobacteria belonging to the group of denitrifying bacteria with diverse metabolic abilities (Dados et al., 2015). As reported previously, P. otitidis is used for wastewater treatment (Dahiya & Mohan, 2016), including contaminant removal processes, such as dye degradation and bioremediation in hydrocarbon-contaminated soils (Dados et al., 2015;Wu et al., 2009).
Vogesella perlucida is a Gram-negative bacteria and was firstly isolated from water samples from springs in Taiwan (Chou et al., 2008). Phylogenetic analysis showed that CNA1 is in the same clade as Acinetobacter johnsonii strain ATCC 17909 T , which indicates a close relationship with Acinetobacter haemolyticus strain ATCC 17906 T . Acinetobacter is a genus of bacteria from the Gamma proteobacteria group, widely distributed in various ecosystems with aerobic denitrification capabilities (Qin et al., 2019). These bacteria are able to reduce nitrate or nitrite to N 2 under aerobic culture conditions (Wang et al., 2020).
Phylogenetic analysis showed that the CNA1 isolate was in the same clade as Acinetobacter johnsonii strain ATCC 17909 T , which indicated a close relationship with Acinetobacter haemolyticus strain ATCC 17906 T (Figure 8). Acinetobacter is a genus widely distributed in various ecosystems with aerobic deni-  Isolation and identification of bacteria removing nitrite ..... (Qoriatul Ilma) trification capabilities (Qin et al., 2019), and can reduce nitrate or nitrite to N 2 under aerobic culture conditions (Wang et al., 2020). In this study, CNA1 isolates reduced nitrite levels, with a low accumulation of ammonium ( Figure 2 and 3), but did not reduce nitrate levels ( Figure 4). K1NA3 metabolizes nitrite and nitrate, as well as forms ammonium. According to Yang et al. (2012), members of Pseudomonas are strong denitrifiers. They have complete denitrification enzymes and can catalyze the reduction of nitrate to nitrogen. These bacteria are able to use oxygen on nitrite or nitrate ions when there is no free oxygen. This aerobic metabolism is shown by its ability to reduce nitrate to ammonium through nitrite (Dahiya & Mohan, 2016). The use of indigenous bacteria in nitrogen-based wastes (ammonium, nitrite, and nitrate) control in aquaculture is still considered relevant. Basically, this waste control is to facilitate the conversion of ammonium to nitrite and/or nitrate to nitrogen gas. Bacteria that decompose ammonium, nitrite, and ni-trate have an important role. By using indicator paper for nitrite or nitrate, the bacteria capable of removing nitrite and or nitrate are Pseudomonas otitidis (K1NA3 and K2NA3), Acinetobacter cumulans (CNA1), and Vogesella perlucida (PRO4NA1). Screening of active bacteria in diminishing nitrogen-based wastes was accelerated by using indicator paper ( Figure 1 and Table 1). By using the spectrophotometer-based method, the bacteria are confirmed to be able to reduce the levels of nitrite ( Figure 2) and nitrate (Figure 4). Diminishing nitrite content was followed by the formation of ammonium (Figure 3). This is due to the dissimilatory nitrite reduction to ammonium (DNRA) process (Stein & Klotz, 2016). Two isolates (K1NA3 and PRONA1) were able to metabolize nitrate ( Figure 4) and producing nitrite ( Figure 5) and ammonium ( Figure 6). No isolate was observed to convert nitrite to nitrate indicating that the oxidation of nitrite to nitrate is not running smoothly. However, DNRA and the oxidation of ammonia to nitrite can be observed in a population. Figure 9. Phylogenetic tree of 16S rRNA sequences of four isolates obtained from bioball. Phylogenetic tree was constructed based on 16S rRNA gene sequencing by the neighbor-joining method. Distances were estimated according to the Kimura two parameter model with bootstrap percentages after 1,000 simulations. The bar represents a 2% estimated sequence divergence. Nitrosomonas europaea strain ATCC 25978 T was used as an out-group.
Indonesian Aquaculture Journal, 17 (1), 2022, 13-22 The performance of isolates in removing ammonium in synthetic wastewater was briefly observed. Axenic cultures of PRONA1 had the lowest DNRA activity, as well as community cultures (CNA1 and PRONA1; and a mixture of K1NA3, K2NA3, CNA1, and PRONA1 should be PRO4NA1) and were able to reduce ammonium in synthetic wastewater after six days of incubation (Figure 7). Ammonium metabolism in mixed bacterial cultures yielded the highest nitrite ( Figure 8). The nitrate content was not detected. The transformation of ammonium, nitrite, and nitrate into nitrogen gas is a complex reaction involving oxidoreductase enzymes of a population or community (Ge et al., 2012;Stein & Klotz, 2016;Zhang et al., 2019). Abiotic environmental factors such as pH, carbon sources, alkalinity, and oxygen could drive the reactions (Li et al., 2016;Dahiya & Mohan, 2016Ge et al., 2012).

CONCLUSIONS
Four bacterial isolates, namely K1NA3 and K2NA3, CNA1, and PRO4NA1, capable of reducing nitrite, nitrate, and ammonium, were successfully isolated from bioballs of a recirculating aquaculture system. Isolates K1NA3 and K2NA3 have the closest relationship to Pseudomonas otitidis; CNA1, and PRO4NA1 have a closer relationship to Acinetobacter cumulans and Vogesella perlucida, respectively. The isolates could be applied to enrich effective bacteria removing nitrogenous compounds in RAS to maintain good water quality and support sustainable fish farming. Further studies are still needed to ensure the role of these bacteria in nitrogenous waste control.