Estuary Dam Suwung is a lagoon formed at the estuary of the Badung River flowing through the city of Denpasar. Badung River has long experienced pollution due to waste disposal from various activities along its bank. The high pollutants load entering the estuary dam of Suwung Denpasar has led to the decreasing quality of the waters and the formation of oxygen deficit zones at the bottom. Pollution containing high concentration of organic materials  tends   to   cause   the   acidic   and  anaerobic conditions of the aquatic sediments. These conditions affect the composition of biological species with biogeochemical cycles. The acidic environment can trigger the formation of reactive metals in the form of their ions, thereby causing metal contamination in the aquatic environment. Meanwhile, sludge from wastewater treatment becomes its own problem with heavy metal content and low pH. This high level contamination of heavy   metal    requires    development     of     a   sludge management system to treat the acidity and heavy metal content. Biotechnology has been utilized in waste water treatment. Using this technology, the contaminant is removed through the precipitation of the contaminant in the reduced form by the microbial activity. Furthermore, on a certain scale, the precipitated contaminants can be recovered.

Anaerobic biological processes encourage sulfide formation by sulfur reducing bacteria. SRB is heterotrophic bacteria that use simple organic compounds as carbon sources. With metabolic respiration, the bacteria utilize sulfate, thiosulfate, sulfites and other reducible sulfur compounds as electron acceptor (Müller et al., 2014).

The sulfur in the oxidized state will be reduced to sulfide in the anaerobic environment (Barton and Faugue, 2009). SRB is a true anaerobic microorganism with a primitive respirative pathway capable of living in extreme environments. The group of bacteria is generally isolated from aquatic sediments that have extreme conditions, as temperature, pH, alkalinity, sulfate, iron, manganese, ammonium and phosphate contents (Rückert, 2016). This research aims to isolate and characterize SRB from water sediments contaminated with domestic waste.

Isolation

The soil samples were taken from Estuary Dam in Suwung, South Denpasar area of Bali. There are about 12 sampling points in one area. For SRB exploration, soil samples taken from sediments were immediately inserted into polyethylene bottles and stored in a cooler box for transporting and then stored in a freezer in the laboratory. The number of samples taken at each sampling point ranged from 100 to 200 g of soil. SRB group isolation was based on Postgate medium B liquid media composition (Atlas, 1993). The pH 4 setting was performed with 10% sulfuric acid and 10% sodium hydroxide prior to sterilization. Samples suspended in 0.85% NaCl and diluted to 10³ were then cultured in an anaerobic tube. The cultures were then incubated at 35°C. Observations were made from the time when color started to change until all turned black. Blacken isolates were then purified in liquid medium (Rückert, 2016). The isolates were suspended and diluted further in the same manner until they reached dilution level of 10¹². Growth time of the bacteria was observed from the time of black color appearance until the entire tube turned black. Isolates grown at the final dilution rate were indicated as cultures with one type of SRB cell.

Characterization of SRB cells

Isolates indicated as SRB cells were tested for their ability to reduce sulfate and increase the pH of the media. Isolates were cultured in a liquid medium containing 500 ppm sulfate. Trials were carried out in media with pH variations of 7, 5, 4 and 3. One mL of SRB cells isolate was transferred aseptically into a threaded tube filled with 1/3 liquid medium (500 ppm sulfate with 7, 5, 4, or 3), and filled up with medium, then incubated at 35°C. On the 15th day, the residual sulfate and pH of the solution were measured. The isolates capable of growing at low pH and reducing sulfate (and increasing pH of the media were selected.

Isolation and characterization

The estuary dam from where the samples were collected has undergone siltation, indicated by its blackish brown sediment, low pH (5, 6 and 3) and high pollutant contents. All 12 soil samples isolated with Postgate B liquid media, change from clear to black under anaerobic conditions, which indicate the presence of SRB (Dennis and Julia, 2014). The black color shows the presence of sulfide (reduced sulfur) which is the result of sulfate reduction by SRB (Xu et al., 2013). The growth rate observed by the time required to turn the colour of the suspension black varies widely. The growth time of SRB from the isolated samples is shown in Table 1. The capability of the SRB to reduce sulfate, and therefore increase pH, as measured after 15 days culture, is shown in Table 2. The observation found nine isolates capable of growing at all pH variations, and only three isolates did not grow at pH 3 (Table 2). The pH changes and sulfate reduction efficiency is shown in Table 3. The sulfate reduction efficiency was determined by calculating the percentage of the reduction of the sulfate concentrations. Table 3 shows the sequence of ability to increase pH and sulfate reduction efficiency of nine isolates. High efficiency sulfate reduction is an important factor for sulfide formation associated with pH increase.

Three isolates appear to have a prominent activity compared to the other six isolates. The three isolates namely DPS 1711, DPS 1703 and DPS 1705, are also the ones with highest growth rates (Table 1). Bacterial cells grow rapidly by splitting in the supporting environment. When food is abundant then the term survival of the fittest which implies that, the winning type is the one who can divide the most rapidly. Faster splitting ability allows bacterial populations to  adjust  immediately to changes in the environment (Shiqiang et al., 2014). Microorganisms interact with their environment in a variety of ways. The ability to utilize certain nutrients, producing metabolites that affect other microorganisms and interacting with the physical and chemical environment are the factors that determine the growth of a microorganism. These conditions define the activity and growth character of a species, which is different from the other species (Kato, 2016). The ability to grow at room temperature is a very important factor for the efficient application of SRB isolates in bioreactors. The ten isolates selected in the first selection, were further selected for the purpose of the determination of the reduction efficiency. The tested isolates were cultured on a Postgate B liquid medium containing 1000 ppm sulfate at pH 3 and incubated at room temperature (27 to 30°C).

Measurements and observations of growth time, optical density, pH increase and sulfate reduction different efficiency are shown in Table 4. Measurements of  optical density, pH increase and sulfate reduction efficiency at room temperature are performed after a 21 day incubation period with estimated exponential phases being exceeded. Isolates capable of growing rapidly at room temperature, raising the pH of the medium and reducing the sulfate with high efficiency (that is >80%) are selected isolates. The selected isolates are DPS 1703, DPS 1705 and DPS 1711 (Figure 1). Growth at room temperature and activity on acidic medium (low pH) are very important properties in the applications for treating acidic mine waste and are important factors for the efficiency of the bioreactor performance.

Cell morphology and physiology

Observations under microscope of simple staining, Gram staining, and spores to determine cell morphology were performed  on  all  three  selected  isolates.  Physiological and biochemical characterization were also performed. The results are shown in Table 5 and the photograph of the isolates in Figure 2. Endospores have very high resistance to heat and are not easily damaged by the effects of chemicals, dryness, radiation, acidity, and dormant for a very long time (Kato, 2016). In Figure 2c, the spherical endospores are in contrast to their rod-shaped vegetative cells. The color difference is caused by the absorption of green color of kit by endospora. Based on the observed characteristics shown by DPS 1703, DPS 1705 and DPS 1711 isolates.

The selected bacteria namely (DPS 1711, DPS 1705 and DPS 1703) Sulfate reducing bacteria are identified as D. orientis. These bacteria exhibit wide range temperature and pH tolerance thus could be applied for treating highly polluted waste such as acid mine waste.

The authors have not declared any conflict of interest