DNA Barcoding and Water Quality Analysis of Nitrifying Bacteria in Lebak Lebung Swamp

ABSTRACT


INTRODUCTION
Nitrification will follow the ammonification process, which is a process of reforming organic nitrogen from land waste into ammonium [1,2].The nitrification process will be followed by an increase in organic nitrogen due to the abundance of landfill being a concern of many scientists because the large amount of organic nitrogen will have negative implications on water quality, including eutrophication or increased organic biomass [3][4][5].The nitrification process is played by the bacteria Nitrosomonas sp. which converts ammonia into nitrites [6] and the bacteria Nitrobacter sp. which will oxidize nitrites to nitrates [7,8].This nitrate is harmless to fish life and tends to be used by aquatic plants to grow and develop [9].The influence of Nitrosomonas and Nitrobacter bacteria is very important in reducing nitrite and increasing nitrate in fish farming [10][11][12].The problems that occur in the field are that the nitrification process does not work properly, causing the buildup of ammonia which will result in the death of the fish.
The main cause is the lack of nitrifying bacteria in helping to decompose organic matter found in aquaculture ponds.
Nitrite is found in the waters in very small amounts compared to nitrates, because it is not stable in the presence of oxygen [13].Nitrate is a source of nitrogen for plants which is then converted to protein.Nitrates are very easy to dissolve in water and are stable [14][15][16].Organic nitrogen in waters is sourced from waste disposal from the land including household activities, industry, fertilization and cultivation which triggers an increase in the amount of organic material entering the waters [17][18][19].The nitrification process in aquaculture ponds is very important with the help of Nitrosomonas and Nitrobacter bacteria, the help of these two genera of bacteria can reduce the ammonia content contained in aquaculture ponds and can increase the fish production process [20][21][22].This prompted the need for studies related to the isolation of nitrifying bacteria as probiotic agents to overcome various problems of fish mortality [22].One of the first steps that can be taken is to screen and isolate nitrifying bacteria.
Previous studies have succeeded in isolating nitrifying bacteria from various cultivation media, such as research conducted by Aswiyanti et al. [23] who isolated nitrifying bacteria from tilapia cultivation media and succeeded in obtaining bacterial isolates of the type Klebsiella spp.This was further explained by Hastuti et al. [24] who isolated nitrifying bacteria from mud crab cultivation media using a recirculation system or water flowing continuously and succeeded in obtaining isolates of Pseudomonas stutzerii and Halomonas sp.Currently there is no research that has isolated nitrifying bacteria in swamp waters so it is possible that the bacteria obtained are local bacteria which can later be used as probiotics for fish farming in swamp waters.This is of course very important for the sustainability of cultivation in peat swamp waters.
Isolation, selection, and characterization are important steps to get the desired bacteria.The stages of the process are needed for the type of bacteria obtained has the ability according to the target.The characterization stage was carried out to determine the type of bacteria and its kinship relationship using 16S rRNA sequencing.Taxonomic analysis using 16S and 18S rRNA genes for sequencing data has shown a valid approach in characterizing bacterial communities [25,26].Aquaculture development activities in swamp water or wetlands that are inundated with water at certain periods of time has the problem of contamination from organic matter, and this waste has the potential as a nitrogen source in swamp water.Therefore, research on the isolation and characterization of nitrifying bacteria in swamp water to be identified.By identifying bacteria that have a role as bioremediators, this can become a tool that can be used to determine the condition of a particular body of water.This is because the presence of these bacteria indicates a process of change that occurs naturally in the waters in order to maintain the stability of environmental conditions and is an illustration that there is pollution/waste contained in these waters.

Research place
This research was conducted at the Laboratory of Microbiology at Department of Mathematic and Natural Science, and Laboratory of Aquaculture and Plant Physiology at Department of Agriculture Cultivation Faculty of Agriculture Sriwijaya University.

Sample description
Samples are stored in a watertight and clean container.All tools and materials as well as the laboratory environment have been ensured to be sterile to avoid external contamination.Soil and air samples from the Lebak Lebung Ogan Ilir swamp waters were taken at the same point, namely the edge.Taking soil samples at the edge makes it easier to take the amount of soil compared to taking the middle part of the water.According to Kusmawati [27], soil is one of the growing media of various kinds of animals, microbes, because it has a complex source of nutrients for bacterial growth.Water quality measurements include temperature using thermometer, pH using pH meter, dissolved oxygen using DO meter and ammonia using a spectrophotometer.

Isolation of bacteria candidates
Isolation of nitrifying bacteria using the enrichment culture method was carried out by inoculating 1 gram of the soil sample obtained into each NA (Nutrient Agar) media.The liquid culture containing the isolate was homogenized with a shaker for 7 days or until a color change occurred.The presence of ammonium-oxidizing bacteria is indicated by a change in the color of the media from red to yellow due to changes in the pH of the media due to the oxidation of ammonium to nitrite, while the presence of nitrate-producing bacteria is indicated by changes in the media.color from clear to cloudy [28,29].Next, the bacterial colonies that grow on certain media in petri dishes are taken one by one and planted back in the dish to be purified by incubating at room temperature (27)(28)℃ in anaerobes conditions.

Observation of colony morphology
The pure bacterial isolate was then identified through two observations, namely: a) observing the morphology of the bacterial isolate which was observed including color, cell shape, edges and surface, b) microscopic observation by testing the gram characteristics of the isolate using differential staining and observing through a microscope with 100× magnification to know the gram test reaction [30,31].

DNA extraction
The bacterial DNA extraction process was carried out using PrestoTM Mini gDNA Bacteria Kit (Geneaid Biotech Ltd., City, Country).The sample required 1 × 106 bacterial cell for one extraction.The DNA extraction procedure was carried out in accordance with the manual PrestoTM Mini gDNA Bacteria Kit.

Amplification and electrophoresis
The PCR material was used 25 μl of DNA extraction.Each reaction contains: go taq green 12.5 µl, there are two pairs of 16S rRNA primers namely 63f primer 1 µl, primer 1387r 1 µl, NFW (Nuclease Free Water) 6.5 µl, DNA template 4 µl.DNA amplification is carried out by stages: initiation cycle at 95℃ for 5 minutes, denaturation at 94℃ for 30 seconds, annealing or primary attachment at 50℃ for 1 minute, extension or elongation at 72℃ for 2 minutes in 30 cycles and the final extension at 72℃ for 5 minutes [32].PCR products are electrophoresed through 1% agarose electrophoresis gel. 1 μL of dye loading was mixed with 6 μL of DNA inserted in each well electrophoresis.Electrophoresis was carried out with 75V power for 50 minutes and the results were immersed into TAE 1X which had been added with diamond nucleic acid dye for 30 minutes, then visualized with Gel Doc.The DNA size of the Gel Doc results using a 1 kb marker.The results of the amplification of known DNA sizes using electrophoresis were then sequenced.

Data analysis
Sequences that have been obtained in the form of fasta format are then aligned using MEGA 6.0 software, then sequenced sequences are taken for later BLAST (Basic Local Alignment Search Tool) which is used to determine the homology of a DNA or amino acid sequence with the data contained in Genbank NCBI (National Center for Biotechnology Information) and Barcode of Life.Furthermore, all sequences are aligned for genetic distance and phylogenetic trees.Phylogenetic trees between bacterial species were constructed using the Maximum Likelihood method.

Isolation and characteristics morphology of bacteria candidates
The results of bacterial isolation obtained in the swampy waters of the swamp obtained a total of 10 isolates, but based on the characteristic equation of each isolate only one isolate was selected for further testing because it has similarities in morphological characteristics.The isolate with the following morphological characteristics (Table 1): The tested bacteria were pure isolates of nitrifying bacteria that had the N1 code.The location of sampling of land and water is carried out in 2 places, namely on the right side and the left side of the edge.According to Kusmawati [27], soil is one of the growing media of various types of organisms, one of them, microbes, because it has a complex source of nutrients for bacterial growth.The results of the morphological characteristics have not been able to provide information about the genus from the isolates that have been obtained.Therefore, further testing of these isolates was carried out.

Molecular characteristics of bacterial isolates
The amplification or multiplication of target DNA is intended to increase the amount of target DNA using the PCR method with universal bacterial primers namely 16S rRNA 63F (Forward) and 1387R (Reverse) and the template of DNA extraction of 4 µL nitrifying bacteria can be detected by DNA visualization.DNA visualization is presented in Figure 1.Based on Figure 1 visualization of the results of amplification of 16S rRNA in ± 1300 bp bacterial isolates.According to Marchesi et al. [33] in practice 63f and 1387r primers were more successful than amplimer 27f-1392r pairs and reinforced 16S rRNA genes from a variety of bacteria that were better than others which are usually used for analysis of bacterial communities.Primary 63f and 1387r have been evaluated to amplify the 16S rRNA gene from Domain bacteria.This primary pair is able to amplify the gene with a size of about 1300 base pairs.The visualization process is one way to determine the quality of DNA isolates and ensure the suitability of primers used by looking at the length of base pair (bp) [34].
The sequenced results are then aligned using Mega 6.0 software.After aligning produce a nucleotide base sequence with a length of 1380 bp.Furthermore, isolates were analyzed by BLAST (Basic Local Alignment Search Tool-nucleotide) at the NCBI website (National Center for Biotechnology Information) showed the results of the percentage similarity of samples of nitrifying bacteria with data found in Genbank.BLAST analysis of nucleotide sequences of 16S rRNA gene samples of nitrifying bacteria isolates N1 had the highest percentage of identity, 93% with Indian species Burkholderia cepacia strain NBRAJG97 and Burkholderia sp.strain 172 1492R originating from Estonia.According to Syakti et al. [35] if homology is more than 97% of the same species, between 93% and 97% the genus is the same but different species, and if it is smaller than 93% it is likely a new species.Usually, a method based on this philosophy is known as the distance method [36].Genetic distance was analyzed using Pairwise Distances with the Maximum Composite Likelihood and Substitutions models to included: Transitions + Transversions in MEGA 6.0 and the results of genetic distance analysis of nitrifying bacteria showed that isolates observed in the study were N1 isolates having the lowest genetic distance of 0.004 (0, 4%) with the Burkholderia cepacia strain of NBRAJG97 (EU734821.1)from India.
The level of genetic similarity of a population can be described by genetic distance from individual members of the population.The smaller the genetic distance between individuals in a population, the more uniform the population will be.Conversely the greater the genetic distance of individuals within a population, then the population has increasingly diverse members [37].

Figure 2. Phylogenetic tree of nitrifying bacteria
The kinship of an organism can be described through phylogenetic trees which are constructed based on genetic distance analysis.The phylogenetic tree is a two-dimensional graph that shows the relationship between organisms or population classifications based on the history of their evolution.Phylogenetic trees are constructed through the Mega 6.0 software application using the Neighbor-Joining (NJ) method [38] of the Maximum Composite Likelihood and Substitutions models to included: Transitions + Transversions with 1000× boostrap [39,40].The eubacteria phylogenetic tree is presented in Figure 2.
The results of phylogenetic tree construction showed that nitrifying bacteria formed branches with a scale of 0.02.The phylogenetic tree of nitrifying bacteria showed that the sample of N1 isolate had a 99% bootstrap value with the species Burkholderia cepacia strain NBRAJG97 from India, this was seen in the species forming its own subcluster.Member of genus Burkholderia are common soil inhabitants and that their biogeographic distribution is strongly affected by soil Ph.Burkholderia strains have a competitive advantage in acidic soils but are outcompeted in alkaline soils [41].Burkholderia cepacia can be classified as follows: Divisio Proteobacteria; Betaproteo bacteria class; Order of Burkholderiales; Burkholderiaceae family; Burkholderia genus [42].B. cepacia is still genomovar (one genetic species that is closely related) with Pseudomonas, namely P. kingii, P cepacia, P. multivorans.Burkohlderia cepacia bacteria are heterotrophic nitrifying bacteria that are able to convert nitrite (NO2-) into nitrate (NO3-) which then converts nitrite (NO2-) into NO with the help of the nitrite reductase enzyme which plays a role in reducing nitrite concentrations.Next, the nitrogen that has been formed is further converted into nitrate (NO3-) by the dioxygenase enzyme under aerobic conditions [43][44][45].
Research conducted by Gohar et al. [46] stated that the Burkholderia cepacian type of bacteria showed strong activity against Aeromonas hydrophila, Edwardsiella tarda, and Vibrio ordalli.The results of the rough extraction of these bacteria produce antimicrobial fractions.They are (1) Phenol, (2) Phenol-4-methyl, (3) 3-benzyl-1,4-diaza-2,5-dioxobicyclo (4) hexadecanoic acid ethyl ester, and (5) 1,2 Benzene acid decarboxylated.Further explained by Young et al. [47].which stated that Burkholderia sp.CC-Al74 can increase the level of P utilization and the total P and N content after endophytic colonization.The Burkholderia genus is rich in nitrogen-fixing and phosphate-solubilizing strains [48].Burkholderia produces a structural array of specific metabolites with antifungal and antibacterial properties.In addition, Burkholderia is a relatively untapped resource for the discovery of natural antimicrobial products with diverse cellular targets and has promising potential for clinical, agricultural and fisheries uses [49].Burkholderia β-Proteobacteria is a promising source of NP, development of Burkholderia bacteria as heterologous hosts and application of Burkholderia in industrial-scale NP production [50][51][52][53][54][55].

Water quality
The results of measurement of water quality at the location where sampling is shown in Table 2.
Measurements of water quality observed at the location of sampling included parameters of temperature, pH, DO and ammonia.The measured temperature has a fairly high value, this is because temperature measurements are carried out during the day, which according to Nelson et al. [51] the amount of heat from sunlight entering the waters or its spread is greater so it can affect the media.In general, the swamp waters are somewhat acidic to neutral (pH 4 to neutral) with more acidic tendencies during the dry season [41].DO (mg.L -1 ) 6.0 -6.2 4.
Amonia (mg.L -1 ) 0.05 In general, DO has a tendency that is directly proportional to depth [42].This is caused increasingly towards the base of air diffusion and photosynthetic activity decreases, and oxygen reduction due to decomposition of organic matter in the water base is getting bigger [43].The ammonia content in swamp water is 0.05 mgL -1 .Based on Marbun et al. [55], unionized free ammonia levels should be no more than 0.2 mgL - 1 .Based on the water quality conditions obtained, the conditions obtained are still considered optimal for the growth of fish and bacteria.There are water quality exceptions to bacterial populations because they have a good ability to withstand even extreme environmental conditions.

CONCLUSION
Burkohlderia bacterium identified in the waters of Lebak Lebung Swamp, Ogan Ilir, South Sumatra are known to have a role as nitrifying bacteria.In the future, they can be applied as bioremediators in mass cultivation activities and in their natural conditions can also be bioindicators of the condition of balance in the aquatic environment.

Figure 1 .
Figure 1.Visualization of 16S rRNA gene isolation results from bacterial isolates M= Marker

Table 2 .
Water quality at the location of sampling