Cadmium (Cd) Reduction Bacteria From Northern Coast Sediment Indramayu, Indonesia


 Cadmium (Cd) is one of the metals that contaminates Karangsong and has found several bacteria that can reduce cadmium (Cd). The purposes of this research are to obtain the type of bacteria and obtain an evolutionary relationship of cadmium (Cd) reduction bacteria from sediments in Karangsong Beach, Indramayu. The method used in this research is an exploratory method and the resulting data is analyzed descriptively. The molecular identification method uses 16S rRNA gene which is a universal primer for bacteria. The bacterial sequences obtained were analyzed with GenBank data from the NCBI website. The results showed that two bacterial isolates were identical to the Pseudoalteromonas issachenkonii strain KMM 3549 (Acc. No. NR 025139.1) with 77% identity value and 100% evolutionary relationship value and Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Acc. No. NR 041787.1) with identity values 97% and evolutionary value 99%.


Introduction
Marine pollution has recently become a problem that threatens the earth and continues to be discussed by the international community. The rapid development of industry can have a negative impact on the environment, especially the marine environment. Pollutant wastes can cause large amounts of heavy metal content in seawater; this will have a negative impact on the survival of marine ecosystems and other living things, especially humans 1 . Several sources of heavy metals in marine waters can come from mining, agricultural waste and industrial waste 2 . Wastes produced from industry are generally heavy metals such as Hg, Cd, Cu, Zn and Ni which are included in the category of Hazardous and Toxic Materials (B3) 3 .
Heavy metal pollution has also occurred in the Karangsong Beach area as an area with rapid industrial growth which also has a quite complex industrial waste problem. One of the reasons of the high pollution case in Karangsong is because it has one of the largest sh auction sites (TPI) in West Java, therefore the high activity of shermen who mostly use diesel, gasoline and also disposal of waste oil from PT. Pertamina resulting in a high pollution to its waters. Various studies show that most of the waters in the Karangsong Coast area are contaminated with heavy metals, especially Cd. Efforts to control heavy metals by developing a consortium of indigenized bacteria as a bioremediation agent for Cd metal pollution in waters contaminated with industrial waste in Karangsong Beach, Indramayu Regency, are strategic steps to ght this problem. These bacteria are known to reduce the concentration of Cadmium (Cd) metal as a bioremediation agent. These bacteria can be identi ed by their types and relationships through molecular identi cation using PCR (Polymerase Chain Reaction) techniques using the 16S rRNA gene.
The primer used in the bacterial DNA ampli cation process is the 16S rRNA gene. The use of the 16S rRNA gene is based on the size of about 1550 base pairs and about 500 bases at the end of the sequence is an area called the hypervariable region. This area is the part that differentiates between organisms.
Primers used in sequence ampli cation will recognize conserved regions and amplify hypervariable regions, thereby obtaining sequences that are unique to these organisms 4 . The purpose of this research was to obtain the types of bacteria and the relationship of the cadmium (Cd) reducing blood bankers from the sediments of Karangsong Beach, Indramayu.

Results
Sampling and Water Data. The water conditions of Karangsong Port are very murky, dark brown in color, have a very dense mud texture and a stinging odor. The parameters used to measure the quality of the waters of Karangsong Port are Temperature, DO, pH and salinity. The quality of Karangsong Waters is shown in Table 1. According to the quality standard of KMN-KLH PP No. 1 Year 2010 on the conduct of water pollution control, the value of pH parameter which categorized as normal is 6 -9. The value of DO (dissolved oxygen) obtained is 5.92, dissolved oxygen is one of the important components in water quality. The average salinity value obtained is 29.7 this indicates that the sampling location is in the estuary area where this area has a salinity range between 5 -35 ppt. salinity also has a role in the solubility of heavy metals, the higher the salinity then the higher the content of heavy metals is due to the disruption of the oxidation process of the heavy metals 5 .
Cadmium Metal Concentration Reduction Test (Cd). Reduction of Cadmium (Cd) heavy metal concentrations was done by experimental test using indigenous bacteria from Karangsong Port.
A treatment with the 1.5 mg. L -1 concentration, it was able to lower the Cd concentration to 0.68 mg. L -1 at 48 th hours; the lowest concentration was 0.653 at 6 th hours (Fig. 1a). At a treatment of 1 mg. L -1 concentration, it could lower the Cd concentration to 0.48 mg. L -1 at 48 th hours; the lowest concentration was 0.445 at the 6 th hours. At a treatment where the concentration was 0.5 mg. L -1 , it was able lower the Cd concentration to 0.32 mg. L -1 at 48 hours; with the lowest concentration of 0.271 in the 12 th hours.
The gene that can cause bacteria to be resistant to the heavy metal cadmium is the cad A-operon gene [6][7] so it is thought that the bacteria taken at Karangsong Port have that gene. The bonding of heavy metals in a solution is by the exchange of ions where the ions in the cell wall of microorganisms are replaced by heavy metal ions 8 . The highest Cd reduction weight was 56.47% in C treatment (1.5 mg. L -1 ) after 6 hours of the treatment, meanwhile the lowest Cd reduction weight was 45.8% after 12 hours in the treatment A (0.5 mg. L -1 ) (Fig. 1b). There are several factors that affect the reduction or absorption process of heavy metals, including phenotype factors, biomass factors, and medium factors.
Isolation of Metal Reduction Bacteria Cd. The bacterial samples used were bacteria samples that were not previously given 70% alcohol, so that they were found to be resistant to heavy metals. Bacterial samples were incubated for 48 hours at 37°C and subsequently bacterial colonies were obtained (Fig. 2).
The bacterial morphology identi cation in Table 2 follows the guidelines 9 . The ability of bacteria to form genetically colored dyes has become special markers for this bacteria type 10 . Bacterial puri cation perform after identi cation process. The purpose of bacterial puri cation is to obtain pure isolates that have the same morphological characteristics and gram type. Bacterial puri cation is done twice to ensure that the puri ed bacteria are pure. The results of bacterial puri cation can be seen in Fig. 3 Gram Staining. Gram staining was performed to identify the bacteria into two groups, namely gramnegative bacteria and gram-positive bacteria. The gram staining technique usually use alcohol and iodine, use dyes as well namely crystal violet and safranin. This observation is carried out microscopically using a microscope with a magni cation of 1000x. The results of the gram stain can be seen in Supplementary Table 1.
The results of gram staining shows that the eight pure isolates tested were gram-negative bacteria, these results are in accordance with the statement in previous studies 11 , that gram-negative bacteria are generally more tolerant of the in uence and exposure to heavy metals than gram-positive bacteria, this is because gram negative bacteria have a complex cell structure and which can bind and immobilize most of the heavy metal ions. Based on Supplementary Table 1, the form of bacterial cells obtained is generally in the form of diplobacilli (A1, A4, A6, A7 and A8) due to the shape of the bacterial cell resembles as a stem and forms two chains, meanwhile in isolate codes A2, A3 and A5 the bacterial cells are oval or coccobacillus. This is in accordance with the statement from previous study 12 that 80% of bacteria found in the sea are rod-shaped.
Test Challenge of Bacteria to Cadmium Metal (Cd). Heavy metal challenge test aims to see the resistance of bacteria to heavy metals. According to another study regarding the bacterial resistance test 13 , the test of bacterial isolate resistance refer to obtain bacterial isolates that are resistant to heavy metals. Isolate of the 8 bacterial, merely 5 isolate of bacterial were truly pure bacteria, namely Cd 2, Cd 3, Cd 5, Cd 6 and Cd 7. These bacteria are known to reduce cadmium concentrations from 1.5 mg. L -1 to 0.681 mg. L -1 and bacterial growth from 1 x 10 6 CFU/ml to 19 x 10 6 CFU/ml. Bacterial isolates that were considered pure then subjected to a challenge test to cadmium (Cd) metal.
Optical Density (OD) values were measured at 0 and 18 hours, furthermore most of bacterial growth was seen from the increase in Optical Density (OD) values at 0 and 18 hours. The results of the challenge test are listed in Table 3.  Table 3. Optical Density (OD) Value at t = 0 and t -18 The Optical Density (OD) value shows the level of bacterial density. Based on the results, the change in the Optical Density (OD) value sequentially from highest to lowest were isolate Cd 5 (1.973); isolate Cd 3 (1.931); isolate Cd 7 (1.868); isolate Cd 6 (1.728) and isolate Cd 2 (1.509). The bacterial density that had been altered from the Optical Density (OD) value, then sort from highest to lowest namely isolate Cd 5 (1.58 x 10 9 ); isolate Cd 3 (1.54 x 10 9 ); isolate Cd 7 (1.49 x 10 9 ); isolate Cd 6 (1.38 x 10 9 ) and isolate Cd 2 (1.21 x 10 9 ). The difference in Optical Density (OD) showed the level of bacterial density. Bacterial growth can be seen from the level of turbidity (optical density) which can be read through the resulting absorbance value. The wavelength used is 600 nm, that is the optimal wavelength in reading the density of the bacterial suspension.
The binding of heavy metals by bacteria can be separated into the binding phase and active transport.
The binding phase depends on cell metabolism, namely absorption through the cell wall or external surface, then followed by active transport which depends on cell metabolism. In metabolic processes, heavy metals can accumulate in cell membranes (extracellular) and in the cytoplasm (intracellular) 14 .
The bacterial isolates then selected into 3 bacteria with the highest density level to proceed the identi cation stage of the molecular method using 16S rRNA. Those bacterial isolates are Cd 5, Cd 3, and Cd 7 isolates. DNA Extraction. DNA extraction is an early stage in molecular analysis. The objectives of DNA extraction is for separate nucleic acids from other components of the cell and obtain DNA concentration and purity values before proceeding to the next stage of the process. Good-quality of DNA purity is indicated by the value of protein and RNA contamination in the solution. The results of DNA purity and concentration are listed in Table 4.  Genomic DNA electrophoresis used a 1 kb marker with a band display of 250 -10,000 bp ranges. The results of visualization in Fig. 4 show that the DNA band measuring 1,500 bp (base pair) matches the target. The DNA bands obtained were accordance with the target because the annealing temperature that had been optimized in the PCR program for the DNA ampli cation process was appropriate and running well. The results of electrophoresis of genomic DNA as a whole apart from the visible DNA bands showed a smear but too thin. Smears can be seen in all three isolate samples. Smears can be caused due to the presence of contaminants such as protein or carrying the remaining solution in the isolation process.
According to previous study 18 , the smear could be the residue of the solutions that were still carried during the isolation process or it could also be DNA which was degraded in the isolation process.  Query coverage is the percentage of nucleotide lengths that correspond to the database contained in BLAST. Query coverage is an important parameter because it rates in percent of sequences in the database masked queries, which con rms whether the sample is covered by all sequences. The cover query values of the isolates were 85% and 89%. The acceptable percentage is at least 95%, except for sequences where a lower reading is applied to a minimum of 75% 19 .
The BLAST results of the two isolates had 77% and 97% identity values. A bacterial species can be said to be the same if it has a homology of more than or equal to 97% 20 . According to previous study about DNA sequence analysis 21 and based on 16S rRNA gene sequence data, identity ≤ 97% can be stated that the isolates being compared are in the same genus, identities between 89 -93% indicate different families. The BLAST results on NCBI said that the Cd 7 isolate is a Pseudoalteromonas spiralis strain Te-2-2 because it has an identity value of 97% with Acc. No. NR 114801.1. Cd 3 isolate has an identity value of 77%, it is suspected that this isolate is a new species that is in the same order, namely Alteromonadales.
The e-value of the two isolates is zero respectively. The e-value 0 obtained shows a signi cant alignment, which means that the search for the specimen sequence in this study is identical, namely from the same genus and even at the species level 22 . The higher the e-value indicates the lower the homology level between the sequences, while the lower the E-value indicates the higher the homology level between the sequences. An e-value of 0 (zero) indicates that the two sequences are identical. Phylogeny Analysis. Phylogenetic tree construction aims to see the relationship between the sample organisms based on their evolutionary relationships with the sequence of comparison organisms originating from the NCBI site. Phylogenetic trees were constructed using the neighbour-joining tree method. Phylogenetic trees were statistically tested using the bootstrap method with 1,000 replications. A bootstrap value of 100 to 1,000 replicates is used to estimate the con dence level of a phylogenetic tree 26 . bootstrap analysis with values of 70% or higher indicates a reliable clustering 28 .
The construction of the phylogeny tree shows that the Cd 3 sequence is related to Pseudoalteromonas issachenkonii strain KMM 3549 (Fig. 5a). The phylogeny tree also shows a kinship relationship between the two high sequences with a bootstrap value of 100% and an identity of 77%. This value indicates that out of 1000 reconstructed phylogeny trees, the Cd 3 sequence has a 100% relationship with Pseudoalteromonas issachenkonii strain KMM 3549. On the other hand, the Cd 7 sequence is related to Pseudoalteromonas tetraodonis GFC strain IAM 14160 (Fig. 5b). The phylogeny tree also shows a kinship relationship between the two high sequences with a bootstrap value of 99% and an identity of 97%. This value indicates that from 1000 reconstructed phylogeny trees, the Cd 7 sequence was 99% related to Pseudoalteromonas tetraodonis GFC strain IAM 14160.

Methods
Sampling. Sediment sampling was carried out from Northern Coast Indramayu, Indonesia more accurately at Karangsong Harbor, Indramayu Regency, West Java, Indonesia with coordinates S 06°56'2" E 107°46'38" using a piston core and a shovel. Samples were taken at ports contaminated with heavy metal Cadmium (Cd) in places that were immersed by water. Samples were taken at a depth of approximately 50 cm above sea level.
Cadmium Concentration Reduction Test (Cd). Seawater was ltered with a 0.22 μm lter then sterilized using an autoclave put into a test bottle with a volume of 500 ml, close the bottle so that no air or other intervention disturb the seawater and let it rest. A stock solution of Cd (SO 4 ) with a concentration of 1.5 mgL -1 (treatment A), 1 mgL -1 (treatment B) and 0.5 mgL -1 (treatment C) were added to the seawater in different tubes to pinpoint each treatment's effect on the result. Then the source of bacteria of about 10 mL was added into each of the test medium and homogenized. After that, the medium is then poured into an 80 ml vial bottle quickly and precisely and labeled starting from T 0 or 0 hours, T 2 or 6 hours, T 3 or 12 hours, T 4 or 24 hours, and T 5 or 48 hours. The metabolism of the bacteria was stopped using 96% alcohol every hour.
Calculation of Bacterial Density. The calculation of bacterial density in this study was carried out using the Total Plate Count (TPC) method. The principle of the Total Plate Count (TPC) method is to grow living microbial cells on one or more media so that these cells multiply and form colonies that can be seen directly with naked eyes without using a microscope, and colonies can be counted using colony counter.
Inoculation of Bacteria. Bacterial inoculation is done by mixing the bacterial sample from pore water and then diluting it to 10 -5 . Then carried out the observation of the morphology of bacteria on the growth media (nutrient agar) using the pour plate method.
Gram stain. Gram staining will classify bacteria into two groups, namely gram-positive and negative. The nal result is gram-positive bacteria marked in purple while gram-negative bacteria are marked in pink.
Cadmium Metal Challenge Test (Cd). This challenge test against heavy metals is to see the resistance of bacteria to heavy metals. Isolate of 8 bacterial were taken in one loop, put into 10 mL of liquid medium (Nutrient Broth) containing Cd (SO 4 ) with concentrations of 1, 1.5 and 2 mg. L -1 . Then the Optical Density (OD) value was measured using a spectrophotometer. Bacterial isolates were incubated into the incubator shaker for 18 hours at 37 °C at a speed of 220 rpm. After that, the nal Optical Density (OD) value was measured to determine the change in the density level of each bacteria.
Extraction of bacterial DNA. Extraction of bacterial DNA was done using the phenol chloroform method, but on the homogenization stage (cell lysis stage), reagents from TRIsure was used. The procedure used is based on the bacterial DNA extraction protocol from TRIsure BIOLINE. The stages of DNA extraction include homogenization, separation phase, DNA precipitation, DNA wash, and DNA re-dissolving. Regarding the concentration of cadmium (Cd) reducing bacteria whose types and relationships are known, it is necessary to test on a eld scale so that it can be applied to aquatic ecosystems that are heavily polluted by metal cadmium (Cd). Figure 1 The Culture of cadmium reduction bacteria. The isolation results obtained colonies that grow on the surface of the medium. Those bacteria are categorized as aerobic bacteria. Bacteria puri cation results. There are two differences in the color of bacterial colonies due to the excretion of substances or pigmentation into the medium.

Figure 4
The result of agarose gel electrophoresis is 1% of the ampli ed DNA. The results of the three bacteria that had been extracted by DNA then ampli ed using the Polymerase Chain Reaction (PCR) method for 29 cycles.