**2. Selection of inoculum and medium for consortium development**

The authors of this chapter have developed a consortium from waste water–fed fish pond at East Kolkata Wetland (EKW), India (22° 27′ N 88° 27′ E) [1] in synthetic medium (DSMZ 641) specific for growing SRB under anaerobic condition, which could reduce soluble sulphate from 1600 ppm to discharge level within three and half hours of incubation at room temperature in a packed bed reactor with stable biofilm for sustained treatment of soluble sulphate. The geographical orientation in terms of slope is such that the entire city's (Kolkata's) run off (which includes contribution of acid rain) drains at EKW. However, there is no toxicity reported in these water bodies. Hence, there is high possibility of these water bodies harbouring efficient SRB. The selection of medium for growth, the inoculum for development of the consortium and the subsequent selection pressures were carefully monitored keeping in mind the need to specifically enrich the SRB with minimal non-SRB so as to ensure insignificant dead mass during bioreactor operation, hence developing a tailor-made consortium for this purpose. Its performance was tested for sulphate reduction from modified synthetic medium (DSMZ 641) prepared using tap water and mining effluent.



Sodium azide is also added to prevent the fungal formation.

The following are the modified medium components, which were effective for SRB in terms of nutrient consumption. This medium is far more advantageous in terms of cost and amount of consumption. It is similar to the

aforementioned media composition with a minor change in components such as ammonium chloride—50% of previously mentioned medium, KH2PO4—25% and yeast extract—50%.

#### **Composition of the mining effluent**

KH2PO4 0.5 g Yeast extract 1.0 g Then, a pinch of Resazurin is added, which is used as a redox indicator.

Na2S2O4 0.1 g NaHCO3 1.68 g Lactic acid 12 ml NaOH 4.4 g

HCl (25%; 7.7 M) 10.0 ml FeCl2 × 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 × 4 H2O 0.1 g H3BO3 6.0 mg CoCl2 × 6 H2O 190.0 mg CuCl2 × 2 H2O 2.0 mg NiCl2 × 6 H2O 24.0 mg Na2MoO4 × 2 H2O 36.0 mg Distilled water 990.0 ml

Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine 10.0 mg Thiamine HCl × 2H2O 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg D-Ca-pantothenate 5.0 mg Vitamin B12 0.10 mg P-amino benzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml

Sodium azide is also added to prevent the fungal formation.

The following are the modified medium components, which were effective for SRB in terms of nutrient consumption.

This medium is far more advantageous in terms of cost and amount of consumption. It is similar to the

**Solution C: Trace element solution 1.0 ml** (SL10)

**Solution D: Vitamin solution 10 ml**

**Solution B**

20 Nuclear Material Performance

Sodium (17.5 ppm), potassium (37.3 ppm), manganese (0.03 ppm), nickel (0.026 ppm), magnesium (17.6 ppm), calcium (540 ppm), total carbon (5.893), inorganic carbon (4.477) and total organic carbon (1.419).

Microorganism preferentially gets attached to the surfaces in favourable conditions like moist surface along with the nutrients as a layer called biofilm. Earlier reports indicated the presence of surfaces to stimulate attached bacterial growth under conditions, which are otherwise too dilute to sustain the microbes [22]. The operation was scaled up to 220 litres. The system involved three columns in series of 78, 71 and 71 litres (**Figure 1**). It showed an efficiency of above 50% soluble sulphate (starting from 1600 ppm) reduction within three and half hours under ambient temperature during both batch and continuous modes. The sulphate reduction varied from 65% to 100% within 24 hours. The biofilm could be well sustained in both polypropylene and steel matrix (**Figure 2**) without any maintenance for more than 18 months.

**Figure 1.** 220-litre packed bed bioreactor for soluble sulphate removal with steel and polypropylene immobilization substrate with defined surface area for bacterial biofilm formation.

**Figure 2.** Scanning electron microscopic image of different matrix with and without SRB biofilm. From extreme left, polypropylene matrix (without biofilm), polypropylene matrix (with biofilm), steel matrix (without biofilm), and steel matrix (with biofilm).
