**4. Biofilm formation and sulphate reduction**

Biofilm, a thick layer of microbial cells embedded into secreted extracellular material on inert matrix such as polypropylene or metals, acts as a constant source of inoculum for the system. The method was adopted from Nasipuri et al. [1]. Two millilitre of sample was centrifuged at 10,000 rcf for 10 minutes to pellet down the cells. The supernatant was mixed with 98 ml of distilled water in a 250-ml conical flask. A 5 ml of conditioning mixture containing hydrochloric acid (6%), isopropanol (20%), water (64%) and glycerol (10%) was added to it for proper mixing. The mixture was then put onto a magnetic stirrer at the maximum speed for 1 minute with a pinch of barium chloride. The solution was then allowed to stand for 2 minutes for settling the barium sulphate precipitate. The absorbance was taken at 420 nm for soluble sulphate measurement using a dual-beam spectrophotometer by Agilent Technologies.

Similarly, for measurement of biofilm, the method of Martin [24] was adopted. The biofilmcontaining matrix was firstly stained for 10 minutes with crystal violet. Vigorous washing was done with distilled water to wash away the loosely bound stain. Ninety-five percent of ethanol was then added to remove the bound stain from the biofilm. The absorbance of the removed stain was measured at 620 nm for biofilm thickness measurements using a dual-beam spec‐ trophotometer by Agilent Technologies.

**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

Inoculum optimization was done based on the extent of sulphate reduction following immo‐ bilization of the consortium onto a matrix as per the method of Nasipuri et al. [1]. The inoculum percentage was varied from 2% to 50% (2%, 5%, 10%, 20%, 30%, 40% and 50%) to maximize the sulphate reduction by the system. Optimum sulphate reduction of 70% was obtained with 10% primary inoculum. It implied that with 10% bacterial inoculum maximum metabolic rate was reached, which eventually resulted in a significant sulphate reduction under optimum condition. But further increase in inoculum percentage resulted in no further increase in

Two types of matrices (polypropylene and steel) with uniform surface areas were tested for the purpose as per the method of Nasipuri et al. [2]. The stainless steel and polypropylene raschig rings showed an overall sulphate reduction of 72.05% and 69.59%, respectively. They were equally efficient as immobilization matrix in terms of sulphate reduction under the same set of conditions. The comparable range of efficiency between stainless steel and polypropy‐ lene raschig ring in terms of sulphate reduction might be due to equal lower pressure drop at effective surface areas and same gas velocity. Our data were also supported by the study of Kolev et al. [23]. In this regard, the scanning electron microscopic images were further used to

Biofilm, a thick layer of microbial cells embedded into secreted extracellular material on inert matrix such as polypropylene or metals, acts as a constant source of inoculum for the system. The method was adopted from Nasipuri et al. [1]. Two millilitre of sample was centrifuged at 10,000 rcf for 10 minutes to pellet down the cells. The supernatant was mixed with 98 ml of distilled water in a 250-ml conical flask. A 5 ml of conditioning mixture containing hydrochloric acid (6%), isopropanol (20%), water (64%) and glycerol (10%) was added to it for proper

visualize the dense biofilm formation on both types of matrices (**Figure 2**).

**4. Biofilm formation and sulphate reduction**

**3. Optimization of inoculum percentage and immobilization matrix**

matrix (with biofilm).

22 Nuclear Material Performance

efficiency in terms of sulphate reduction.

The effect of biofilm formation by SRB consortium on sulphate reduction was checked for 90 days. The biofilm thickness (left) compared with sulphate reduction (right) performed by the system for the above-mentioned period (**Figure 3a** and **b**) revealed oscillatory nature of both biofilm formation and associated sulphate reduction. It is an inherent nature of a biofilmbased system. The biofilm thickness was not directly correlated with the extent of reduction. This is because biofilm thickness as reflected by the method of Martin et al. [24] consists of active cells, inactive cells and the extracellular polymeric substances, although the reduction is due to the function of just the active cells. Hence, both show an oscillatory pattern but are not directly dependent on one another. The evidence of an oscillatory nature in biofilms was also observed by others [25, 26], which eventually supported the former statement.

**Figure 3.** Graphical representation of biofilm formation by the SRB consortium on matrix (left) with associated sul‐ phate reduction (right).
