**2. Materials and methods**

All the species of cyanide family (viz. thiocyanate, cyanide and their metal complexes) have potential to interacting with living cells and strong tendencies to connect to proteins and thereby acts as a non-competitive inhibitor (Westley, 1981). This fact necessitate the

basis before its discharge in soil and water environment; as it may pose detrimental im‐ plications on aquatic life. Moreover, in water scarce situations such untreated and partial‐ ly treated wastewaters could not be recycled back into the industrial processes. The

range of 5 - 110 mg/l (Mudder and Whitlock, 1984). Although many statutory agencies across the world have set the statutory limits for cyanide and heavy metal discharge, till

free cyanide species. The US-health service cites 0.01 mg/l as guideline and 0.2 mg/l as the permissible limit for cyanide species. In India, the Central Pollution Control Board (CPCB) had set a Minimum National Standard (MINAS) limit for cyanide as 0.2 mg/l. Therefore, the cyanide bearing effluents generated from industries needs suitable treat‐ ment to bring down the total cyanide levels below 0.2 mg/l. Taking into consideration the

be on the safer side. In order to minimize the risk of exposure to the public and aquatic

and Kulkarni (2008) have reported the environmental sensitivity and safety aspects in mining industries in regard to cyanide. Impact of cyanide species on fresh water fish Cat‐

Numerous technologies are currently employed to detoxify SCN- bearing effluents; and the most widely being used is direct alkaline chlorination or addition of hypochlorite. However, this method produces large aggregates of chemical sludge, which does not have any further utilization and is environmentally hazardous to handle (Lanza and Bertazzoli, 2002). As per Indian regulations, such hazardous chemical sludge is transited from the industrial location to a specially designed Treatment, Storage and Disposal Facility (TSDF) thereby increasing the overall energy consumption, transportation cost and air pollution. Secondly, chlorina‐

limits especially when heavy metals are present in the effluents. Thirdly, chlorination in‐ creases the total dissolved solids (TDS) content of the treated wastewaters, which makes it unfit for further use. Other physico-chemical processes like hydrogen peroxide oxidation, ozone oxidation, electrolytic decomposition, etc. are highly expensive and are rarely used

Bioremediation (biological treatment system) using metabolically active (live) microorgan‐ ism is one such effective alternative for the detoxification of toxic chemical wastes. This process has immense potential of treating variety of pollutants (both toxic and non-toxic); has several advantages over conventional methods and therefore being explored by the re‐ searchers world-wide. Microorganisms capable of utilizing C-1 compounds like CN-

tive treatment process capable of achieving high degradation efficiency at low cost.

(and other CN-

. Thus, there is a pressing need for the development of an alterna‐

date there are no such prescribed limits set or documented for SCN-

to adequately detoxify the effluents on priority

is approximately 7 to 10 times less toxic then

could readily be deduced to 1 mg/l to

species) within the statutory

and

contaminated wastewaters is therefore necessary. Patil

discharge. Earlier sci‐

arising from all the above mentioned sources is normally in the

industries using and/or emanating SCN-

32 Applied Bioremediation - Active and Passive Approaches

entific studies indicate that in general, SCN-

mentioned facts, standards for discharge of SCN-

tion also fails to bring the concentration of SCN-

for the treatment of SCN-

la catla have also been reported (Prashanth and Patil, 2008).

ecosystems, the clean-up of SCN-

concentration of SCN-

#### **2.1. Analyses, chemicals and glassware**

Potassium SCN- (KSCN) was obtained from Qualigens, Mumbai, India. SCN- assay was car‐ ried out spectrophotometrically (Spectronic, Model-20D, India) using ferric nitrate method at 460 nm as described in Standard Methods (APHA-AWWA-WEF, 1998). Digital pH meter (Elico, Model Ll-120, India) was employed to determine pH of solutions. Bacterial popula‐ tion from culture media, activated sludge and soil were determined microscopically (Met‐ zer, India, METZ-778A) using Neubauer's chamber (Fein-Optik, Blankenburg, GDR) and by total viable count (TVC). Analytical grade chemicals were used for all experiments. Re‐ agents were prepared in glass distilled water and stored under refrigerated conditions (8-10°C).

#### **2.2. Enrichment and isolation of SCN degrading bacterial consortium**

Enrichment culture and growth of mixed bacterial community (bacterial consortium) was carried out using M-9 minimal salts medium (MSM) (Patil and Paknikar, 2000a). One litre of medium contained Na2HPO4.2H2O - 3.0 g; KH2PO4 - 1.5 g; NaCl - 0.25 g; distilled water - 1000 ml and 1 ml/l trace metal solution (Bauchop and Elsden, 1960; Millar, 1972). The medi‐ um pH was adjusted to 7.5 using 1 M NaOH/HCl. Glucose (10 mM) was added as the sole source of carbon and energy. SCN- (50 mg/l) was supplemented to the enrichment medium as the sole source of nitrogen. Enrichment culture for the isolation of SCN degrading micro‐ organisms were set-up in aerobic and unsterilized conditions using activated sludge (ob‐ tained from secondary treatment of sewage treatment plant) and garden soil. Both the samples were collected in clean polythene bags and carried to the laboratory. Two cylindri‐ cal glass jars (reactors) of 1200 ml capacity each were employed for the enrichment purpose. Working volume of the glass reactor was 1000 ml. 100 ml of activated sludge or 100 g of gar‐ den soil was added in 900 ml M-9 MSM containing SCN and glucose as the source of nitro‐ gen and carbon, respectively in order to obtain the final concentration of SCN and glucose as 50 mg/l and 10 mM, respectively. Enrichment was carried out at the pH 7.5. Both the glass reactors were incubated at room temperature (30±2°C). Air was sparged continuously at the bottom of medium at the rate of 1000±50 ml/min using electrical aerator units. Seven to eight successive transfers of 10% solution enriched with bacterial flora were given periodically in the fresh M-9 MSM containing SCN and glucose as mentioned earlier (Patil, 2006; Patil, 2008a).

Where, C = SCN-

stant (h-1); Co= initial SCN-

**2.5. Utilization of SCN-**

**2.6. Factors influencing SCN-**

parameters on SCN-

**2.7. Biosorption of SCN-**

concentration.

**2.8. Impact of cations and anions on SCN-**

SCN-

(0.1-1 mM).

(Remi, CIS-24 BL) at 150 rpm for 48-72 h (Patil, 2011).

temperature (20 – 45 °C), initial cell density (105

concentration (mg/l); t = time (h); k = rate of reaction / first order rate con‐

 **as the sole source of cellular nitrogen by bacterial consortium**

biodegradation. Experimental conditions used were as follows: 150 ml

= SCN-

Development of a Bioremediation Technology for the Removal of Thiocyanate from…

concentration at time t.

http://dx.doi.org/10.5772/56975

35

(50 mg/l) and glucose (10

cells/ml) and glucose (1-20 mM) were

cells/ml was used

was

concentration (mg/l) and Ct

 **biodegradation**

mM). 1 ml of previously grown culture (for 24 h) having cell density of 108

ing the others constant. Periodic analyses were conducted as mentioned earlier.

capacity Erlenmeyer flasks with 25 ml M-9 MSM containing SCN-

Batch mode experiments on SCN- biodegradation were conducted in aseptic conditions with 100 ml M-9 MSM in 250 ml conical flasks. The medium was augmented with carbon and ni‐ trogen sources with different combinations as mentioned: - (i) potassium thiocyanate - KSCN (50 mg/l) as the sole carbon and nitrogen source or (ii) KSCN (50 mg/l) and glucose (10 mM) as the sole nitrogen and carbon, respectively or (iii) KSCN (50 mg/l) and NH4Cl (1 mM) as the sole carbon and nitrogen, respectively or (iv) KSCN (50 mg/l), NH4Cl (1 mM) and glucose (10 mM) as the nitrogen, nitrogen and carbon sources, respectively. All experi‐ ments were conducted at pH 7.0. Flasks were incubated at 30°C on a rotary shaker incubator

Series of batch culture experiments were conducted to investigate the influence of various

as inoculum. The flasks were incubated in stationary conditions. Impact of pH (5.0 - 9.0),

checked by running different set of experiments, wherein, one parameter was varied keep‐

 **by bacterial consortium at high cell density**

Experiments were performed in 150 ml flasks. 50 ml aliquots of SCN- (50 mg/l) adjusted to optimum pH (7.0) was contacted with bacterial consortium (108 cells/ml). The culture was inactivated by boiling for the period of 10 min prior to contact. The flasks were incubated at 30°C on a rotary shaker (150 rpm) for 24 h. Bacterial cells were separated by centrifugation at 1000 rpm for 10 min and the cell free supernatant was subjected to determine the residual

Batch experiments were performed under optimized conditions as described earlier. Impact of diverse cations, especially heavy metals (0.1 mM each) on biodegradation of SCN-

studied. Metals were added as sulfate salts (range 0.1-1 mM). To study the influence of sul‐ fates, additional sulfate was added to the medium as sodium sulfate. Chlorides were added as sodium chloride in the range of 1-10 mM, while cyanide was added as sodium cyanide

– 109

 **biodegradation**

#### **2.3. Purification and Identification of bacterial cultures**

The enrichment cultures as obtained were streaked on nutrient agar medium and M-9 agar medium (containing SCN and glucose) plates in aseptic conditions at 35°C for 48-96 h. In all, six diverse types of bacterial colonies (three each from garden soil and activated sludge enrichment) appeared on the plates. The cultures were further purified and then transferred to nutrient agar and M-9 agar slopes. By way of periodic transfers, one set of bacterial con‐ sortium was consistently maintained in liquid medium (i.e. M-9 MSM) (Patil 2008a; Patil, 2008b). Further, the isolated bacterial cultures were subjected for microscopic examination (Gram staining and motility) and cultural characteristics on the nutrient agar plates. Ber‐ gey's Manual of Systematic Bacteriology (Holt, 1989) was used to identify the SCN- degrad‐ ing bacterial cultures up to genus level only.

#### **2.4. SCN degradation efficiency of the isolated bacterial cultures**

Quantitative studies on SCN degradation were performed to determine the efficiency of isolated cultures in their individual capacity and mixed form. Experiments were per‐ formed in 250 ml Erlenmeyer flasks containing 100 ml sterile M-9 MSM (pH 7.0) and 50 mg/L potassium thiocyanate (KSCN), which acted as a nitrogen source. 10 mM glucose was used as carbon source. Bacterial cell suspension of 0.1 ml containing 108 cells/ml were inoculated into the flasks and were incubated at 30°C in a rotary shaker incubator (Remi, India) at 150 rpm for 48 h. Requisite controls were used and experiments were per‐ formed in duplicates and repeated twice. SCN contents were determined periodically as mentioned earlier. SCN degradation efficiency of individual and mixed bacterial culture was expressed in terms of percent total SCN degraded in 48 h. Reaction rate and first or‐ der rate constant for SCN biodegradation were calculated experimentally using equation 1 and 2, respectively (Sellers, 1999).

$$
\Delta \mathbf{C} / \Delta \mathbf{t} = \mathbf{k} \ \mathbf{C} \tag{1}
$$

$$\left(\ln \mathbf{C}\_t - \ln \mathbf{C}\_o = -\mathbf{k} \,\mathrm{t}\,\mu\right) \tag{2}$$

Where, C = SCN concentration (mg/l); t = time (h); k = rate of reaction / first order rate con‐ stant (h-1); Co= initial SCN concentration (mg/l) and Ct = SCN concentration at time t.

#### **2.5. Utilization of SCN as the sole source of cellular nitrogen by bacterial consortium**

Batch mode experiments on SCN- biodegradation were conducted in aseptic conditions with 100 ml M-9 MSM in 250 ml conical flasks. The medium was augmented with carbon and ni‐ trogen sources with different combinations as mentioned: - (i) potassium thiocyanate - KSCN (50 mg/l) as the sole carbon and nitrogen source or (ii) KSCN (50 mg/l) and glucose (10 mM) as the sole nitrogen and carbon, respectively or (iii) KSCN (50 mg/l) and NH4Cl (1 mM) as the sole carbon and nitrogen, respectively or (iv) KSCN (50 mg/l), NH4Cl (1 mM) and glucose (10 mM) as the nitrogen, nitrogen and carbon sources, respectively. All experi‐ ments were conducted at pH 7.0. Flasks were incubated at 30°C on a rotary shaker incubator (Remi, CIS-24 BL) at 150 rpm for 48-72 h (Patil, 2011).

#### **2.6. Factors influencing SCN biodegradation**

samples were collected in clean polythene bags and carried to the laboratory. Two cylindri‐ cal glass jars (reactors) of 1200 ml capacity each were employed for the enrichment purpose. Working volume of the glass reactor was 1000 ml. 100 ml of activated sludge or 100 g of gar‐

as 50 mg/l and 10 mM, respectively. Enrichment was carried out at the pH 7.5. Both the glass reactors were incubated at room temperature (30±2°C). Air was sparged continuously at the bottom of medium at the rate of 1000±50 ml/min using electrical aerator units. Seven to eight successive transfers of 10% solution enriched with bacterial flora were given periodically in

The enrichment cultures as obtained were streaked on nutrient agar medium and M-9 agar

all, six diverse types of bacterial colonies (three each from garden soil and activated sludge enrichment) appeared on the plates. The cultures were further purified and then transferred to nutrient agar and M-9 agar slopes. By way of periodic transfers, one set of bacterial con‐ sortium was consistently maintained in liquid medium (i.e. M-9 MSM) (Patil 2008a; Patil, 2008b). Further, the isolated bacterial cultures were subjected for microscopic examination (Gram staining and motility) and cultural characteristics on the nutrient agar plates. Ber‐ gey's Manual of Systematic Bacteriology (Holt, 1989) was used to identify the SCN- degrad‐

isolated cultures in their individual capacity and mixed form. Experiments were per‐ formed in 250 ml Erlenmeyer flasks containing 100 ml sterile M-9 MSM (pH 7.0) and 50 mg/L potassium thiocyanate (KSCN), which acted as a nitrogen source. 10 mM glucose was used as carbon source. Bacterial cell suspension of 0.1 ml containing 108 cells/ml were inoculated into the flasks and were incubated at 30°C in a rotary shaker incubator (Remi, India) at 150 rpm for 48 h. Requisite controls were used and experiments were per‐

 **degradation efficiency of the isolated bacterial cultures**

gen and carbon, respectively in order to obtain the final concentration of SCN-

and glucose as the source of nitro‐

and glucose as mentioned earlier (Patil, 2006; Patil,

and glucose) plates in aseptic conditions at 35°C for 48-96 h. In

degradation were performed to determine the efficiency of

degradation efficiency of individual and mixed bacterial culture

biodegradation were calculated experimentally using equation

ΔC / Δt = k C (1)

ln Ct – ln Co =-kt μ (2)

contents were determined periodically as

degraded in 48 h. Reaction rate and first or‐

and glucose

den soil was added in 900 ml M-9 MSM containing SCN-

**2.3. Purification and Identification of bacterial cultures**

the fresh M-9 MSM containing SCN-

34 Applied Bioremediation - Active and Passive Approaches

ing bacterial cultures up to genus level only.

formed in duplicates and repeated twice. SCN-

was expressed in terms of percent total SCN-

medium (containing SCN-

Quantitative studies on SCN-

mentioned earlier. SCN-

der rate constant for SCN-

1 and 2, respectively (Sellers, 1999).

2008a).

**2.4. SCN-**

Series of batch culture experiments were conducted to investigate the influence of various parameters on SCN biodegradation. Experimental conditions used were as follows: 150 ml capacity Erlenmeyer flasks with 25 ml M-9 MSM containing SCN- (50 mg/l) and glucose (10 mM). 1 ml of previously grown culture (for 24 h) having cell density of 108 cells/ml was used as inoculum. The flasks were incubated in stationary conditions. Impact of pH (5.0 - 9.0), temperature (20 – 45 °C), initial cell density (105 – 109 cells/ml) and glucose (1-20 mM) were checked by running different set of experiments, wherein, one parameter was varied keep‐ ing the others constant. Periodic analyses were conducted as mentioned earlier.

#### **2.7. Biosorption of SCN by bacterial consortium at high cell density**

Experiments were performed in 150 ml flasks. 50 ml aliquots of SCN- (50 mg/l) adjusted to optimum pH (7.0) was contacted with bacterial consortium (108 cells/ml). The culture was inactivated by boiling for the period of 10 min prior to contact. The flasks were incubated at 30°C on a rotary shaker (150 rpm) for 24 h. Bacterial cells were separated by centrifugation at 1000 rpm for 10 min and the cell free supernatant was subjected to determine the residual SCN concentration.

#### **2.8. Impact of cations and anions on SCN biodegradation**

Batch experiments were performed under optimized conditions as described earlier. Impact of diverse cations, especially heavy metals (0.1 mM each) on biodegradation of SCN was studied. Metals were added as sulfate salts (range 0.1-1 mM). To study the influence of sul‐ fates, additional sulfate was added to the medium as sodium sulfate. Chlorides were added as sodium chloride in the range of 1-10 mM, while cyanide was added as sodium cyanide (0.1-1 mM).

#### **2.9. Degradation of SCN from industrial effluent by bacterial consortium**

SCN effluent was synthetically prepared in the laboratory because of the difficulty in pro‐ curement of effluent from industry. This was to test the practical applicability of the micro‐ bial process for degradation of SCN- . Batch experiments were performed as mentioned earlier under optimized conditions (pH 7.0, temperature 30°C and bacterial cell density of 108 cells/ml). Thiocyanate served as nitrogen source, while sucrose (COD 500 mg/l) was used as carbon source. Parameters such as pH, SCN- , COD and soluble metal content were meas‐ ured at regular intervals for a period of 48 h.

#### **2.10. Treatment of SCN waste in a Continuous Treatment System (CTS)**

Thiocyanate containing simulated was treated in a continuous treatment system (CTS) as shown in Fig. 1. The CTS comprised of cylindrical glass column (height, 24 cm; diameter, 8 cm and total volume 0.2 L) containing one-litre simulated SCN- effluent (50 mg/l SCN- ) hav‐ ing COD of 500-600 mg/l. The consortium culture was inoculated at the level of 108 cells/ml (final cell density) and the contents of the reactor were stirred by sparging air at the rate of 1000 ml/min. The pH of wastewater supplemented with nutrients was adjusted between 7.0-7.3 (using 1 M NaOH/H3PO4) and then added from the top of the reactor by manual ad‐ justment at the flow rate of approximate 40-50 ml/h as calculated from mass balance equa‐ tion. The treated effluent was removed from the bottom at the same flow rate. The CTS was operated at ambient temperature (30±2°C) in continuous mode for over a period of 30 days (720 h) by periodically checking the influent and effluent water characteristics for pH, SCN- , COD and cell count according to the method prescribed in Standard Methods (APHA- AW‐ WA-WEF, 1998).
