IDENTIFICATION OF BACTERIA FROM POST TIN MINING POND AND THEIR ABILITY TO FORM BIOFILMS AT DIFFERENT PH

A number of water quality indicators in the tin post-mined pond of a certain age indicate that the water condition is acidic, low dissolved oxygen content, low cation exchange capacity, and polluted by heavy metals. Restoration of the water quality of post-tin mining pond can use microorganisms as bioremediation agents. Microorganisms live by forming microbial community structures called biofilms. The aims of this study was to identify and find out the optimal pH of biofilm formation biofilm-forming bacteria from post-tin mining pond. The steps of research method was the isolation of bacteria by the spread plate technique, the biofilm formation test by the crystal violet technique, and the identification of bacteria macroscopically, microscopically, and physiologically. The isolation results showed that the highest bacterial density was at station 3 with a total of 8.1x103 cfu/ml. The results of the visualization of biofilm formation find out the A8 isolate at pH 5 with the most concentrated staining, while the highest Optical Density (OD) value for each pH was 0.11245 (pH 3) for A8 bacteria, 0.1901 (pH 5) for I1 bacteria and 0.1901 (pH 5) for A8 bacteria of 0.08945 (pH 7). There were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium 08945 (pH 7). Based on identification results, biofilm-forming bacterial isolates from the tin-mining pond of Rebo Village there were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium 08945 (pH 7). There were 14 isolated bacterial belonging to the Genus Branhamella, Bacteroides, Aeromonas, Bacillus and Clostridium.


INTRODUCTION
Continuous exploration of mineral resources in Bangka Belitung in the form of tin (Sn) has an impact on environmental damage. One of the consequences of these exploration activities is the formation of excavated soil in the form of a lake which is well-known as Kolong (pond) in Bangka Belitung community (Triswiyana et al., 2019). Based on Suryadin (2011), kolong (pond) is interpreted as stagnant water caused by tin sand excavation carried out by the miners. The formed of pond started from the excavation of tin sand, then was filled with water so that the volume of water increased and filled the pond made by the miners.
After tin mining the pond has a lot of potential water to be utilized, but it has not been carried out optimally, both for primary needs and secondary activities such as for agriculture, fisheries, and animal husbandry (Suryadin, 2011;Prasetiyono, 2015). The lack of utilization is due to the certain age, the water quality shows poor conditions, namely the acid pond water reaches a pH < 4.0 which is part of Acid Mining Water (AAT), low DO levels, low value of cation exchange capacity, and contaminated by heavy metals such as Cr, Cu, and Pb (Kurniawan, 2017;Kurniawan & Mustikasari, 2019).
The effort to restore the pond water condition after tin mining with a bioremediation approach is very potential alternative because of the low cost. In an effort to explore potential microbes for  (Donlan & Costerton, 2002). The biofilm matrix has the potential to be a biological agent that can be used to reduce water pollutants in the form of heavy metals such as Cr (VI), Cu²⁺ and Pb²⁺ (Kurniawan et al., 2018; (Merina et al., 2011;Gao et al., 2012;Julistiono et al., 2018).

This research was conducted from
November 2020

Sampling
Samples were taken from post-tin mining water in the form of aquatic plants as much as the size of sterile plastic.
Previously, prepared sterile plastic as a sample container. Samples were taken using tweezers by clamping and shaking in order to dry. Then the sample was put in sterile plastic and labeled.

Using Pour Plate Technique
The dilution process was carried out from pond water plants to a dilution of 10-6. Aquatic plants that had been taken from the location were placed in sterile petri dishes containing sterile under water.
Samples of aquatic plants were rubbed and 1 ml of each sample was taken, put into an Erlenrmeyer flask containing 9 ml of sterile bottom water, homogenized and obtained a dilution of 10-1. Then from the 10-1 dilution, 1 ml of water was taken using a micropipette, which was put into 9 ml of sterile distilled water and obtained a 10-2 dilution. Dilution to obtain a dilution of 10-3, 10-4 and 10-5 (Azizah et al., 2017). The results of the dilution in the test tube were isolated into NA media using the pour plate method and repeated 3 times.

Data analysis
Data were analyzed descriptively and inferentially. Species data, biophile absorbance values were displayed in tabular or graphic form. Different tests were carried out to determine the best pH for biofilm formation.

Water Parameter Measurement
The

Biofilm-forming Bacteria
The activity of biofilm-forming bacteria from bacterial colonies was observed qualitatively using the crystal violet technique on a microplate.
Microplate containing bacterial isolates after washing will produce a thick color if it is a biofilm-forming bacteria. The result of visualization of bacterial isolate staining shows a crystal violet color as shown in Figure 4. Bacteria that can form biofilms and cannot form biofilms can be seen in Figure   5. Adhesion to the bottom of the plate becomes the basis for determining whether or not a bacterial biofilm was formed. In

Identification of bacterial isolates
Bacterial isolates growing at the three research points were identified manually The results of bacterial isolation at  (Table 3).
Bacteroides Aeromonas Aeromonas Furthermore, at Point 3, more isolates were obtained, namely 8 bacterial isolates. The identification of the eight bacterial isolates showed that there was a genus Aeromonas (2), Bacillus (2),

Bacillus Clostridium
The results of the identification in Table 2, Table 3 and Table 4 obtained the results of point 1 with 3 bacterial isolates of the genus Branhamella (Table 2), Point 2 obtained 3 bacterial isolates in the form of Genus Bacteroides and Aeromonas (Table   3) and Point 3 are Genus Aeromonas (2), Bacillus (2), Bacteroides (2) and Clostridium (2) ( Table 4). The bacteria of this genus were found because they were bacteria that were tolerant to metal polluted environments. Yulistia et al., (2015) explained that the isolates of coal ash waste obtained were 3 genera of bacteria, Clostridium showed that the best biofilmforming potential with the highest (biofilm parameters) was at pH 7.