Ammonium removal characteristics of facultative heterotrophic nitrifying bacteria isolated from shortfin eel (Anguilla bicolor) recirculation aquaculture system

Nitrifying bacteria play a role in reducing the amount of toxic ammonia in the rearing media. The purpose of this study was to characterize, and identify nitrifying bacteria isolated from shortfin eel (Anguilla bicolor) recirculation aquaculture system. The selective anorganic nitrifying medium was used to isolate the bacteria, followed by cultivation of the bacterium in the organic medium (Triptone Soya Agar). Bacterial characterization was carried out for nine days using a nitrification activity test. A safety test was conducted by intraperitoneal injection of bacteria in healthy eels (Anguilla bicolor). Phenotypic characterizations and analysis of the 16S rRNA and DNA gyrB genes were used to identify the bacteria. This study resulted in the successful isolation of five isolates. The three isolates (ENS2, ENS4, and ENS5) were non-pathogenic and exhibited high nitrifying activities. The isolates reduced 40-50% of ammonia concentration in three days, resulting in a 2-5 mg/L of nitrate concentration in the medium. The 16S rRNA and gyrase B genes sequences of ENS5 were close to Klebsiella quasipneumoniae, but with the low similarity (97.9% and 99.3%, respectively). It is concluded that recirculation aquaculture system of shortfin eel (Anguilla bicolor) harbor of facultative heterotrophic nitrifying bacteria has potential for further application in shortfin eel aquaculture waste treatment facilities.


Introduction
Aquaculture is one of the alternatives to fulfilling fish demands for human consumption [1].Indonesian aquaculture production is increasing at least 12 million tons from 2015 to 2019 [2].Shortfin eel (Anguilla bicolor) is one of important aquaculture products of Indonesia that has a high selling value both in the domestic and international markets [3].The development of eel culture is struggling with the slow growth rate [4].Several rearing technologies were developed to increase production by optimizing feeding and environmental conditions.High feeding rate has been resulting the enrichment of organic matter in the rearing media that affects water quality [5].Decomposition of the feed waste produce toxic nitrogen compounds such as ammonia and nitrite.1260 (2023) 012047 IOP Publishing doi:10.1088/1755-1315/1260/1/012047 2 An alternative that has been developed to manage the negative effects of the intensive rearing system is the application of recirculation systems [6], the system that works by circulating water through a biofilter to remove dirt and feed residues with a reuse of 90% of the water [7].The RAS system able to control fish waste including feces and feed waste to reduce toxic ammonia content in rearing water [5].The ammonia reduction process in RAS system is mainly occurred in biofilter [5,8].Nitrifying bacteria oxidize ammonia to nitrites or oxidize nitrites to nitrates [5].Nitrifying bacteria can be isolated from aquaculture water [9].The addition of nitrifying bacteria in an effort to improve the quality of fish farming water has proven effective in reducing ammonia content to minimize fish mortality due to ammonia poisoning [10].Therefore, this study was carried out to isolate, identify and examine ammonium removal characteristics of facultative heterotrophic nitrifying bacteria from shortfin eel (Anguilla bicolor) recirculation aquaculture system.

Materials and methods
The present research was approved by ethical clearance commission at Universitas Gadjah Mada, Indonesia with approval number of 00016/04/LPPT/V/2021.

Water sampling and quality measurement
Samples were taken from eel rearing media with a recirculation system at the Aquaculture Laboratory, Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, Indonesia.Samples were taken from the surface of the media that contained the accumulation of eel faces and the culture media column.Samples were taken from culture tanks and stored in 50 ml falcon tubes.At the same time, measurements of pH, temperature, and DO levels of the culture media were carried out.

Bacterial isolation and purification
Nitrifying bacteria was isolated by dilution of the water and spread on top of the nitrification medium [9] [11].The inoculated medium was then incubated for 24 hours at 30 qC.Then purification of the separated colonies was carried out by re-culturing on nitrification medium [12].The purified colonies on agar nitrification medium were incubated for 24 hours.After purification, the bacteria were then stored in 30% glycerol TSB medium and then stored in the freezer at -25 qC and 3% trehalose TSB and then stored in the freezer at -80 o C.

Non-pathogenicity test
The non-pathogenicity test was carried out by injection of pure isolate on elver-sized eels (3 fish each) measuring 12-14 cm at a dose of 10 6 cells/ml/fish [13].Pure bacterial isolates were cultured on liquid medium and injected intraperitoneally into 3 test fish as much as 0.1 ml/fish and the mortality of the test eel was observed for 7 days.Pathogenic bacteria will cause death in test fish, while non-pathogenic bacteria will not cause death in fish and can be used in the next stage of research.

Nitrification activity test
The nitrification test was carried out based on previous study [14], bacteria were cultured on liquid nitrification medium and incubated for three days.The content of ammonia, nitrite, and nitrate in the liquid nitrification medium was monitored every day to determine the ability of each isolate to convert ammonia to nitrite and nitrite to nitrate.The concentration of ammonia, nitrite and nitrate were determined by using API kit with spectrophotometric method.A standard curve was made from absorbances of standard solutions for ammonia, nitrite, and nitrate [15][16][17].

Bacterial Identification
Bacteria were identified based on PCR, sequencing and identification based on the BLAST (Basic Local Alignment Search Tool) in NCBI of the 16S rRNA and DNA gyrase B genes [14] [18].Phenotypic characterization was also conducted to understand the bacterial physiological characteristics according to our previous study [13,14].

Water Quality, bacterial isolation and characterization
Bacterial sample were collected from the eel tanks with water temperature of 28 qC, dissolved oxygen of 4.4 mg/L and pH of 7.7.Five pure colonies, namely ENS2, ENS4, ENS5, ENS8, and ENS 10 were collected in this study.The bacteria have different morphological characteristics, especially on their sizes.Five isolates had a milky white color with a glossy surface and entire edges.ENS4, ENS5, and ENS10 isolates had medium colony sizes and ENS2 and ENS8 isolates had small colony sizes.The bacteria were then stored in 30% glycerol TSB medium and then stored in the freezer at -25 q C and 3% trehalose TSB and then stored in the freezer at -80 qC.

Non-pathogenicity test
Survival rate of the experimental fish during challenge infection is presented in table 1.The five bacteria obtained fish survival rate of 67-100 %.Survival rate of ENS2, ENS4, ENS5, and control were 100%, while ENS8 and ENS10 isolates were 67%.During the non-pathogenicity test observation period, ENS2, ENS4, and ENS5 isolates did not show any signs of illness in the fish and the response to feeding was good, while the ENS8 and ENS10 isolates showed signs of hemorrhage on the dead eels [19].

3.3.Reduction of Ammonia
Based on the figure 1, ENS4 and ENS5 isolates decreased the amount of ammonia in the nitrification medium from day three to nine, while ENS2 isolates fluctuated.The best nitrification activity in reducing ammonia was achieved by ENS5 isolate, which reduced ammonia from 1.21 mg/L to 0.588 mg/L in nine days.This indicates that ENS5 isolates have the potential to be applied as nitrifying bacteria in rearing media.

Accumulation of Nitrate
Based on the figure 3, the three isolates, ENS2, ENS4, and ENS5 had the highest nitrifying activity in producing nitrate on the third days, ranging from 4.63 to 5.46 mg/L.The highest nitrate accumulator was ENS5 isolate, which produced a peak nitrate on the third day of 5.46 mg/L.

Phylogenetic tree of 16S rRNA and DNA gyrase B
The 16SrRNA and DNA Gyrase B of ENS5 were successfully amplified and used for sequencing analysis (Figure 4).The result of BLAST using the 16S rRNA gene showed that the isolates had similiarities above 97% to ENS5 isolate was Klebsiella quasipneumoniae subsp.similipneumoniae strain 2437, K. pneumoniae strain TERI-Chilika-07, Klebsiella pneumoniae strain B17, Klebsiella sp.

Discussion
This study isolated five of nitrifying bacteria from the recirculating system of eel (Anguilla bicolor) rearing media.Bacterial isolation was carried out using a selective nitrification medium [23,24].Isolation of five nitrifying bacteria in the present study was associated with good water quality for eel's rearing based on optimum range of water quality [25].Microorganisms could play important role for improvement of water quality [26].The use of nitrifying bacteria as microorganisms in the nitrification process reduced the toxic compound ammonia (NH3) to a less toxic compound, namely nitrate [8].Pathogenic bacteria can cause infectious diseases which often cause mass fish death in a short time [27].Meanwhile, three bacterial isolates, namely ENS2, ENS4, and ENS5 provide no fish mortality or disease symptoms in eel.Hence, we conclude that the three bacteria were non-pathogenic.
Nitrifying bacteria in the present study were the member of heterotrophic bacteria.This study is different to previous report that ammonium oxidizing bacteria usually are Nitrosomonas sp., Nitrosococcus sp., and Nitrosospira sp., the nitrite oxidizing bacteria could be Nitrospira sp. or Nitrobacter sp.[28].The ENS5 isolate in this study was identified as genus Klebsiella based on the closest relationship based on the 16S rRNA and DNA gyrase B genes.Klebsiella could be an opportunistic pathogen that is a common cause of infection in humans and animals, although some nonpathogenic Klebsiella also existed in the environment [29].Further genomic characterization of the bacteria is important to confirm the identity up to species level.
Morphological and biochemical test on the ENS5 isolate showed that the bacteria were Gram negative, non-motile, showed a positive reaction in the catalase, oxidase test, and produced gas.The genus Klebsiella is a gram negative, facultative anaerobic, short rod-shaped and non-motile [31].The ENS5 was fermentative (according to OF test) and showed a negative response to the ornithine, indole and H 2S decarboxylation tests.The ENS5 isolate could ferment lactose, sucrose, and glucose.These characters were similar to the characters of the member of Klebsiella Genus.
Klebsiella sp.ENS5 had the highest rate of ammonia oxidation and nitrate production.Under aerobic conditions, ammonia will go through a mineralization process to be nitrite and nitrate [32].The ENS5 isolate able to reduce ammonia in nitrification medium from 1.21 mg/L to 0.63 mg/L within three days and continued to oxidize the ammonia for a period of nine days.ENS5 isolate had high nitrifying activity in producing nitrate due to the high activity of these bacteria in converting ammonia to nitrate.This is shown by Andriani [33], that high levels of ammonia and nitrite can be caused by the presence or absence 1260 (2023) 012047 IOP Publishing doi:10.1088/1755-1315/1260/1/0120477 of activity from bacteria that can help the process of ammonia oxidation and nitration.Based on the nitrification activity test, the isolate selected for molecular identification was ENS5 isolate.
Results of the present study is similar to other study in Tilapia [14] which succeeded in isolating Klebsiella sp. with strong nitrification activity.Klebsiella sp. is a heterotrophic bacterium that can remove ammonia quickly and can utilize ammonia as an energy source to multiply cells [34].It is also demonstrated that also explained that the species Klebsiella variicola has the ability for heterotrophic nitrification and aerobic denitrification [35].Klebsiella penumoniae has nitrification activity where the bacteria able to reduce ammonia from 27 ppm to 5 ppm in 48 hours post-incubation, and 22 ppm to 11 ppm in 72 hours post-incubation [36].Klebsiella pneumoniae is closely related to K. quasipenumoniae that usually found in sediments or aquatic environments [37].Nitrifying bacteria perform aerobic oxidation of ammonia and nitrites during the nitrification process.
There are several enzymes that participate in the oxidation of reduced nitrogen compounds.Ammonia oxidizing bacteria, oxidize ammonia with the help of an integral membrane enzyme, namely ammonia monooxygenase (amo) and a periplasmic membrane enzyme, namely hydroxylamine oxidoreductase.Through the help of integral membrane enzymes with such ammonia monooxygenase (amo) encodings, ammonia (NH 3) is converted into NH2OH and H2O.Periplasmic membrane with a hydroxylamine oxidoreductase bather, plays a role in oxidizing NH2OH to nitrites (NO2) [38].Klebsiella has been reported to have express ammonia monooxygenase (amo), nitric oxide reductase (nor), nitrate reductase (nar), nitrite reductase (nir), nitrous oxide reductase (nos) genes [34].With these genes, Klebsiella could convert toxic ammonia into nitrate [38].Klebsiella sp.ENS5 of the present study is potential candidates for nitrifying bacteria that can be applied in aquaculture.

Conclusion
Five nitrifying bacteria were successfully isolated from recirculating aquaculture system of eel media.ENS5 isolate has the highest nitrifying activity i.e reduce 50% of the total ammonia concentration in three days, resulting a 2-5 mg/L of nitrate concentration in the medium.The 16S rRNA and DNA gyrase B genes sequences of ENS5 were closes to Klebsiella quasipneumoniae, but with the low similarity (97.9% and 99.3%, respectively).

Figure 1 .
Figure 1.Amount of ammonia in test medium of the present study during nine days of examination.

4 3. 4 .
Accumulation of NitriteBas0ed on the figure2, each isolate ENS2, ENS4, and ENS5 increased until the third day which was 0.336 mg/L; 0.202 mg/L; and 0.267 mg/L.The concentration of nitrite in the nitrification medium of the three isolates decreased on the ninth day.

Figure 2 .
Figure 2. Amount of nitrite in test medium of the present study.

Figure 3 .
Figure 3. Amount of nitrate in test medium during nine days of examination in this study.

Figure 4 .
Figure 4.The 16S rRNA (a) and DNA Gyrase B (b) of ENS5 isolate in the present study.

Table 1 .
The survival rate of the non-pathogenicity test on the test fish.

Table 2 .Table 2 .
[20]otypic characters of selected nitrifying bacteria Biochemical test was used to determine the characteristics of organisms and compared with the results of molecular identification[20].Observations of the biochemical test of ENS5 isolate are presented in Biochemical characteristics of ENS5 isolate.