**3. Results and discussion**

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

Both the enrichment culture (garden soil and activated sludge) elucidated that the time in‐ curred reduced significantly with each subsequent transfer cycle for complete disappear‐ ance of SCN- . Time taken for biodegradation in first, third and fifth cycle was 100, 80 and 70 hours, respectively. After seventh cycle the time taken for biodegradation of >98% SCN was stabilized around 40-45 hours. Each subsequent transfer was given in fresh M-9 MSM soon after the SCN concentration reached to < 1 mg/l (efficiency ≥98%) in the previous cycle. The bacterial count was consistently >108 cells/ml during each transfer cycle. pH and total viable count (TVC) of garden soil prior to enrichment was 8.12 and 3.5 x 108 cells/ml, respectively; and for activated sludge it was 7.64 and >1.2 x 1010 cells/ml, respectively.

of microorganims. There are reports of successfully utilizing this tool for the isolation of mi‐ croorganism capable of degrading toxic and hazardous chemicals like SCN- and MxCN (Pa‐ til, 1999; Patil, 2008a; Sorokin *et al*., 2001). Reduction in time during each subsequent transfer could be explained by the fact that bacterial flora in the enrichment medium got gradually acclimatized to the hazardous chemical environment. High bacterial population (>108 cells/ml) in both the procured samples indicated the presence of substantial organic matter content and nutrient availability, thus giving enhanced probability to obtain SCN- degrad‐

**Figure 1.** Schematic outline of laboratory scale Continuous Treatment System (CTS) for degradation of thiocyanate

In all, six heterotrophic bacterial cultures (three each from both enrichment cultures) capable

plate and spread plate technique that were employed for purification of cultures. Microscop‐ ic examination showed that all the six bacterial culture were Gram-negative rods and motile. Detailed cultural characteristics were previously reported by Patil (2008a). Based on cultural

were isolated by enrichment technique subsequently followed by streak

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

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

37

ing cultures.

of degrading SCN-

**3.2. Purification and identification of bacterial cultures**

The main objective of the present work was to isolate bacterial cultures capable of degrading SCN from the aqueous industrial wastes. In order to accomplished this objective, activated sludge and garden soil was subjected to the most powerful tool called 'enrichment culture', which is popularly being used world across by the microbiologists to selected desired type

**Figure 1.** Schematic outline of laboratory scale Continuous Treatment System (CTS) for degradation of thiocyanate

of microorganims. There are reports of successfully utilizing this tool for the isolation of mi‐ croorganism capable of degrading toxic and hazardous chemicals like SCN- and MxCN (Pa‐ til, 1999; Patil, 2008a; Sorokin *et al*., 2001). Reduction in time during each subsequent transfer could be explained by the fact that bacterial flora in the enrichment medium got gradually acclimatized to the hazardous chemical environment. High bacterial population (>108 cells/ml) in both the procured samples indicated the presence of substantial organic matter content and nutrient availability, thus giving enhanced probability to obtain SCN- degrad‐ ing cultures.

#### **3.2. Purification and identification of bacterial cultures**

**2.9. Degradation of SCN-**

**2.10. Treatment of SCN-**

WA-WEF, 1998).

ance of SCN-

after the SCN-

SCN-

**3. Results and discussion**

**3.1. Enrichment and isolation of SCN-**

bial process for degradation of SCN-

36 Applied Bioremediation - Active and Passive Approaches

as carbon source. Parameters such as pH, SCN-

ured at regular intervals for a period of 48 h.

SCN-

 **from industrial effluent by bacterial consortium**

. Batch experiments were performed as mentioned

, COD and soluble metal content were meas‐

) hav‐

,

was

cells/ml

 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‐

 **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-

(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‐

Both the enrichment culture (garden soil and activated sludge) elucidated that the time in‐ curred reduced significantly with each subsequent transfer cycle for complete disappear‐

stabilized around 40-45 hours. Each subsequent transfer was given in fresh M-9 MSM soon

bacterial count was consistently >108 cells/ml during each transfer cycle. pH and total viable count (TVC) of garden soil prior to enrichment was 8.12 and 3.5 x 108 cells/ml, respectively;

The main objective of the present work was to isolate bacterial cultures capable of degrading

 from the aqueous industrial wastes. In order to accomplished this objective, activated sludge and garden soil was subjected to the most powerful tool called 'enrichment culture', which is popularly being used world across by the microbiologists to selected desired type

hours, respectively. After seventh cycle the time taken for biodegradation of >98% SCN-

and for activated sludge it was 7.64 and >1.2 x 1010 cells/ml, respectively.

 **degrading bacterial consortium**

. Time taken for biodegradation in first, third and fifth cycle was 100, 80 and 70

concentration reached to < 1 mg/l (efficiency ≥98%) in the previous cycle. The

ing COD of 500-600 mg/l. The consortium culture was inoculated at the level of 108

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

> In all, six heterotrophic bacterial cultures (three each from both enrichment cultures) capable of degrading SCN were isolated by enrichment technique subsequently followed by streak plate and spread plate technique that were employed for purification of cultures. Microscop‐ ic examination showed that all the six bacterial culture were Gram-negative rods and motile. Detailed cultural characteristics were previously reported by Patil (2008a). Based on cultural

and biochemical characteristics, all the six identified bacterial cultures belonged to the genus *Pseudomonas* as and reported by Patil (2008b).

of bacteria were efficient compared to individual (pure) isolates (Table 1). These results con‐ firmed the studies carried out by Patil and Paknikar (2000a) on biodegradation of copperand zinc-cyanide using bacterial consortium. In this study, the consortium consisted of four bacterial isolates out of which three were *Pseudomonas* sp. and one was *Citrobacter* sp. The

study could be a manifestation of the natural diversity. Bacterial consortium isolated from activated sludge was more efficient than the consortium isolated from garden soil. Uninocu‐

Experimental determination of reaction rate and first order rate constants are essentially needed because such data gives valuable information regarding the time requirement for re‐ action completion and the size of the treatment facilities that must be provided (Patil, 2008b;

This experiment was carried out only on bacterial consortium isolated from activated sludge because it was more efficient than the consortium isolated from garden soil. Detailed results of this experiment could be obtained from Patil (2011). Overall results are summarized as

remained unchanged throughout the tested period of 48 h. However, it was found that the

the presence external carbon source like glucose (10 mM) within 40 h with an efficiency of >99.9%. This was also confirmed from the control experiments run simultaneously. In the

external nitrogen like ammonium chloride resulted in complete cessation of consortium

carbon (glucose) and nitrogen (ammonium chloride) source, showed an interesting diauxic growth (diauxie) pattern of the bacterial consortium as shown in Fig. 2. The bacterial consor‐ tium preferentially utilized ammonium chloride first until its depletion and only then

All bioremediation processes essentially depends on the availability of principal nutrients in the wastes that could potentially be utilized by the microorganisms as either carbon and/or

um as both carbon and nitrogen source, then at practical scale external supplementation of nutrients will not be required, thereby benefiting the industries economically those using

growth utilizing it as suitable growth substrate. It is well known that concentration of nitro‐ gen required for a given amount of growth is less than the requirement for carbon it might be easier for bacterial consortium to utilize SCN- as the source of nitrogen in the presence of a separate source of carbon and energy (Patil, 1999). Therefore, enrichment culture was de‐

as nitrogen source.

degradation efficiency among the cultures tested in the present

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

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

when supplemented as the sole carbon source in the presence of

levels confirmed that biodegradation of

was supplemented in M-9 MSM as the sole car‐

. The SCN- concentration of 50 mg/l

as the sole source of cellular nitrogen in

was supplied in MSM along with external

is utilized by bacterial consorti‐

, cyanide and metal-cyanides. How‐

compound posed toxic problems to the consortium for

degradation by the cultures.

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

39

wide variation in SCN-

SCN-

Sellers, 1999).

**3.4. Utilization of SCN-**

third combination, SCN-

lated controls did not show any decrease in SCN-

follows. It was established that when SCN-

bon and nitrogen, the consortium failed to utilize SCN-

bacterial consortium was capable of utilizing SCN-

growth. In the fourth combination, when SCN-

nitrogen source. Elucidating this is crucial because if SCN-

microbial technologies for the effluents containing SCN-

switched over to the utilization of SCN-

ever, in the present study, the SCN-

was the predominant reaction taking place during SCN-

The microbial source employed for enrichment culture for the isolation of thiocyante de‐ grading microorganisms were garden soil and sewage sludge of STP. These sites did not have any past history of cyanide or SCN contamination. The prime objective was to test whether SCN degrading bacterial cultures could be isolated from such non-cotaminated sites and secondly to conduct a comparative assessment of the cultures isolated from two completely different niche areas. The fact that six SCN- degrading cultures could be isolated from these samples indicates that SCN- degradation is an intrinsic property of certain micro‐ organisms and that no prior exposure is required to induce this property. SCN degrading ability of various heterotrophic and autotrophic microorganisms have been reported by few authors (Kwon *et al*., 2002; Patil, 2006; Sorokin *et al*., 2001; Stratford *et al*., 1994).

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

Data in Table 1 depicts the wide variation of SCN- degradation efficiency of the bacterial iso‐ lates. However, the bacterial consortium isolated from garden soil and activated sludge showed maximum degradation of SCN- (>99.9%) in 42 and 36 h giving the SCN degradation rate constant (k) of 0.0931 and 0.1086 per h, respectively. In contrast, isolate-2 degraded only 75.9% of SCN- in 48 h (k = 0.029 per h). It was also observed that the first order rate constant of bacterial consortiums was 2-3 folds higher than their individual isolates.


**Table 1.** SCN degradation efficiency of pure and mixed bacterial cultures (Conditions: pH 7.0; Temperature 30°C; Inoculum size 105 cells/ml; SCN conc. 50 mg/L; Glucose 10 mM; Agitation speed: 150 rpm; Incubation time: 48 h) (Patil, 2008b)

This experiment was conducted to ascertain the efficacy of bacterial cultures in their individ‐ ual capacity and in consortium form. And the results clearly revealed that that consortium of bacteria were efficient compared to individual (pure) isolates (Table 1). These results con‐ firmed the studies carried out by Patil and Paknikar (2000a) on biodegradation of copperand zinc-cyanide using bacterial consortium. In this study, the consortium consisted of four bacterial isolates out of which three were *Pseudomonas* sp. and one was *Citrobacter* sp. The wide variation in SCN degradation efficiency among the cultures tested in the present study could be a manifestation of the natural diversity. Bacterial consortium isolated from activated sludge was more efficient than the consortium isolated from garden soil. Uninocu‐ lated controls did not show any decrease in SCN levels confirmed that biodegradation of SCN was the predominant reaction taking place during SCN degradation by the cultures. Experimental determination of reaction rate and first order rate constants are essentially needed because such data gives valuable information regarding the time requirement for re‐ action completion and the size of the treatment facilities that must be provided (Patil, 2008b; Sellers, 1999).

and biochemical characteristics, all the six identified bacterial cultures belonged to the genus

The microbial source employed for enrichment culture for the isolation of thiocyante de‐ grading microorganisms were garden soil and sewage sludge of STP. These sites did not

sites and secondly to conduct a comparative assessment of the cultures isolated from two completely different niche areas. The fact that six SCN- degrading cultures could be isolated from these samples indicates that SCN- degradation is an intrinsic property of certain micro‐

ability of various heterotrophic and autotrophic microorganisms have been reported by few

Data in Table 1 depicts the wide variation of SCN- degradation efficiency of the bacterial iso‐ lates. However, the bacterial consortium isolated from garden soil and activated sludge

rate constant (k) of 0.0931 and 0.1086 per h, respectively. In contrast, isolate-2 degraded only 75.9% of SCN- in 48 h (k = 0.029 per h). It was also observed that the first order rate constant

**With culture Control**

**(without culture)**

*Pseudomonas* sp. # 1 78.24 0 0.815 0.0317 *Pseudomonas* sp. # 2 75.91 0 0.790 0.0297 *Pseudomonas* sp. # 3 82.47 0 0.859 0.0362

*Pseudomonas* sp. # 4 92.07 0 0.959 0.0528 *Pseudomonas* sp. # 5 87.65 0 0.913 0.0435 *Pseudomonas* sp. # 6 89.41 0 0.931 0.0467

degradation efficiency of pure and mixed bacterial cultures (Conditions: pH 7.0; Temperature 30°C;

This experiment was conducted to ascertain the efficacy of bacterial cultures in their individ‐ ual capacity and in consortium form. And the results clearly revealed that that consortium

conc. 50 mg/L; Glucose 10 mM; Agitation speed: 150 rpm; Incubation time: 48 h)

 **degradation Rate of Reaction**

**(mg/l/h)**

0 1.189 0.0931

0 1.387 0.1086

organisms and that no prior exposure is required to induce this property. SCN-

authors (Kwon *et al*., 2002; Patil, 2006; Sorokin *et al*., 2001; Stratford *et al*., 1994).

showed maximum degradation of SCN- (>99.9%) in 42 and 36 h giving the SCN-

>99.9 (in 42 h)

>99.9 (in 36 h)

of bacterial consortiums was 2-3 folds higher than their individual isolates.

 **degradation efficiency of the isolated bacterial cultures**

degrading bacterial cultures could be isolated from such non-cotaminated

contamination. The prime objective was to test

degrading

degradation

**First Order Rate constant (per h)**

*Pseudomonas* as and reported by Patil (2008b).

38 Applied Bioremediation - Active and Passive Approaches

have any past history of cyanide or SCN-

**Source Bacterial Isolates % SCN-**

Bacterial consortium

Bacterial consortium

(1+2+3)

(4+5+6)

whether SCN-

**3.3. SCN-**

Bacterial cultures isolated from garden soil

Bacterial cultures isolated from activated sludge

**Table 1.** SCN-

(Patil, 2008b)

Inoculum size 105 cells/ml; SCN-

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

This experiment was carried out only on bacterial consortium isolated from activated sludge because it was more efficient than the consortium isolated from garden soil. Detailed results of this experiment could be obtained from Patil (2011). Overall results are summarized as follows. It was established that when SCN was supplemented in M-9 MSM as the sole car‐ bon and nitrogen, the consortium failed to utilize SCN- . The SCN- concentration of 50 mg/l remained unchanged throughout the tested period of 48 h. However, it was found that the bacterial consortium was capable of utilizing SCN as the sole source of cellular nitrogen in the presence external carbon source like glucose (10 mM) within 40 h with an efficiency of >99.9%. This was also confirmed from the control experiments run simultaneously. In the third combination, SCN when supplemented as the sole carbon source in the presence of external nitrogen like ammonium chloride resulted in complete cessation of consortium growth. In the fourth combination, when SCN was supplied in MSM along with external carbon (glucose) and nitrogen (ammonium chloride) source, showed an interesting diauxic growth (diauxie) pattern of the bacterial consortium as shown in Fig. 2. The bacterial consor‐ tium preferentially utilized ammonium chloride first until its depletion and only then switched over to the utilization of SCN as nitrogen source.

All bioremediation processes essentially depends on the availability of principal nutrients in the wastes that could potentially be utilized by the microorganisms as either carbon and/or nitrogen source. Elucidating this is crucial because if SCN is utilized by bacterial consorti‐ um as both carbon and nitrogen source, then at practical scale external supplementation of nutrients will not be required, thereby benefiting the industries economically those using microbial technologies for the effluents containing SCN- , cyanide and metal-cyanides. How‐ ever, in the present study, the SCN compound posed toxic problems to the consortium for growth utilizing it as suitable growth substrate. It is well known that concentration of nitro‐ gen required for a given amount of growth is less than the requirement for carbon it might be easier for bacterial consortium to utilize SCN- as the source of nitrogen in the presence of a separate source of carbon and energy (Patil, 1999). Therefore, enrichment culture was de‐

dation. This suggests that SCN-

**3.4. Factors influencing SCN-**

Factors influencing SCN-

maximum SCN-

In general, the SCN-

ents containing SCN-

SCN-

SCN-

revealed that biodegradation of SCN-

utilization by consortium culture is inducible. It was also

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

growth after the exhaustion of ammonium chloride from medium in the first phase. The bio‐ mass that built-up in the first phase of growth was readily available in the medium for SCNdegradation in the second phase, which ultimately led to the rapid biodegradation of SCN-

This result immediately suggests its possible application in bioreactor designing that will re‐ tain large microbial biomass. Immobilization of the biomass in bioreactor using inert materi‐

in the MSM was concomitant with the increase in bacterial population. The fact that the final

tised SCN- tolerant culture, having high SCN- removal efficiency and therefore has immense potential of using the microbial technology on industrial scale. The treatment of wastewater involves a number of chemical and biological reactions and conversions. The rate at which these reactions and conversions occur decides the size of the treatment facilities that must be

by the bacterial consortium isolated from activated sludge was comparatively more efficient than the consortium isolated from garden soil. This might be due to the acclimation/toler‐ ance of sewage microorganisms to a variety of hazardous and non-hazardous waste contam‐ inants/components naturally existing in it, thereby making them more tolerant and efficient

from activated sludge because of its high degradation efficiency compared to the consorti‐ um isolated from garden soil as mentioned earlier. Table 2 shows that degradation of SCNwas significantly influenced by the various factors tested. Optimum pH and temperature for

the optimized conditions of pH and temperature, the initial cell density had a substantial in‐

um occurred in wide range of pH (6.0-9.0), while the optimum being 7.0. From practical applicability point of view very little or no pH adjustment would be required for the efflu‐

 biodegradation. This may be perhaps due to the formation of ammonia as one of the by-products of SCN- degradation, which neutralized the accumulated carboxylic acids in the medium. These results corroborate with the studies carried out by other researchers on the

 degradation process completed within 24 h with >99.9% efficiency. As regard to car‐ bon source, the consortium culture exhibited maximum biodegradation efficiency only

biodegradation was restricted to the bacterial consortium isolated

. With initial cell density of 108

biodegradation (>99.9%) was found to be 7.0 and 30°C, respectively. Under

containing effluents released from various industries have pH in neu‐

. Experiments also showed the unchanged pH of the solution after

al will certainly hasten the process of biodegradation of toxic SCN-

cell density obtained was considerably high (>108

fluence on the biodegradation efficiency of SCN-

tral to alkaline range. Our study showed that growth of SCN-

above glucose concentration of 5 mM.

Further, it could be also observed from the experiments that decrease of SCN-

provided (Tchobanoglous and Burton, 1997). The study also showed that SCN-

degraders as compared to the microbial flora prevailing in garden soil.

 **biodegradation**

took place rapidly (within 25 h) in second phase of

.

cells/ml) indicated the use of well acclima‐

.

41

concentration

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

degradation

cells/ml,

degrading bacterial consorti‐

**Figure 2.** Diauxic growth pattern exhibited by bacterial consortium in the presence two nitrogen sources (viz. SCNand ammonium chloride) in the presence external carbon source (glucose). Growth of consortium (■) and SCN degra‐ dation (▲) in the presence of two N sources; SCN concentration in absence of consortium (●); Cessation of bacterial growth in absence of either nitrogen or carbon source (◆) (Patil, 2011)

signed/manipulated for the isolation of microorganisms capable of degrading SCN as the source of nitrogen nitrogen (Patil, 2006; Patil, 2008a). The experiments conducted explicitly proved that SCN is used by the consortium as nitrogen source in the presence of external carbon viz. glucose, thereby giving the C/N ratio of 10. In view of microbial process devel‐ opment, it is imperative to supplement some cheaper source of carbon like molasses, which is readily available in India at cheaper rate. Patil (1999) had successfully demonstrated the use of molasses as carbon source to develop a microbial technology for metal cyanide biode‐ gradation/removal from wastes utilising it as the sole nitrogen source. There are few reports, which describe microbial SCN degradation utilising it as the sole nitrogen source (Bipinraj *et al.* 2003; Patil, 2011; Sorokin *et al.* 2001). The bacterial consortium ceased to grow when SCN was supplied as sole carbon source in the presence of external nitrogen. This could be attributed to the higher amount of available nitrogen compared to carbon (C/N ratio 0.5). Obviously the culture would find it more difficult to obtain sufficient amount of energy from low amount of carbon.

Example of diauxie pattern (biphasic growth) in *Escherichia coli* in the presence of two car‐ bon sources (viz. glucose and lactose) is well documented (Atlas, 1997). In the present study, diauxic growth pattern was observed when two nitrogen salts (i.e. SCN and ammonium chloride) along with one carbon source (glucose) were supplied to the consortium. Ammoni‐ um chloride acted as preferred growth substrate by the consortium followed by SCN degra‐ dation. This suggests that SCN utilization by consortium culture is inducible. It was also revealed that biodegradation of SCN took place rapidly (within 25 h) in second phase of growth after the exhaustion of ammonium chloride from medium in the first phase. The bio‐ mass that built-up in the first phase of growth was readily available in the medium for SCNdegradation in the second phase, which ultimately led to the rapid biodegradation of SCN- . This result immediately suggests its possible application in bioreactor designing that will re‐ tain large microbial biomass. Immobilization of the biomass in bioreactor using inert materi‐ al will certainly hasten the process of biodegradation of toxic SCN- .

Further, it could be also observed from the experiments that decrease of SCN concentration in the MSM was concomitant with the increase in bacterial population. The fact that the final cell density obtained was considerably high (>108 cells/ml) indicated the use of well acclima‐ tised SCN- tolerant culture, having high SCN- removal efficiency and therefore has immense potential of using the microbial technology on industrial scale. The treatment of wastewater involves a number of chemical and biological reactions and conversions. The rate at which these reactions and conversions occur decides the size of the treatment facilities that must be provided (Tchobanoglous and Burton, 1997). The study also showed that SCN degradation by the bacterial consortium isolated from activated sludge was comparatively more efficient than the consortium isolated from garden soil. This might be due to the acclimation/toler‐ ance of sewage microorganisms to a variety of hazardous and non-hazardous waste contam‐ inants/components naturally existing in it, thereby making them more tolerant and efficient degraders as compared to the microbial flora prevailing in garden soil.

#### **3.4. Factors influencing SCN biodegradation**

signed/manipulated for the isolation of microorganisms capable of degrading SCN-

proved that SCN-

SCN-

which describe microbial SCN-

0

dation (▲) in the presence of two N sources; SCN-

growth in absence of either nitrogen or carbon source (◆) (Patil, 2011)

10

20

30

**Thiocyanate Conc. (mg/L)**

40

50

60

40 Applied Bioremediation - Active and Passive Approaches

from low amount of carbon.

source of nitrogen nitrogen (Patil, 2006; Patil, 2008a). The experiments conducted explicitly

**Figure 2.** Diauxic growth pattern exhibited by bacterial consortium in the presence two nitrogen sources (viz. SCNand ammonium chloride) in the presence external carbon source (glucose). Growth of consortium (■) and SCN-

0 10 20 30 40 50 60 70 **Time (h)**

carbon viz. glucose, thereby giving the C/N ratio of 10. In view of microbial process devel‐ opment, it is imperative to supplement some cheaper source of carbon like molasses, which is readily available in India at cheaper rate. Patil (1999) had successfully demonstrated the use of molasses as carbon source to develop a microbial technology for metal cyanide biode‐ gradation/removal from wastes utilising it as the sole nitrogen source. There are few reports,

*et al.* 2003; Patil, 2011; Sorokin *et al.* 2001). The bacterial consortium ceased to grow when

Example of diauxie pattern (biphasic growth) in *Escherichia coli* in the presence of two car‐ bon sources (viz. glucose and lactose) is well documented (Atlas, 1997). In the present study,

chloride) along with one carbon source (glucose) were supplied to the consortium. Ammoni‐ um chloride acted as preferred growth substrate by the consortium followed by SCN-

diauxic growth pattern was observed when two nitrogen salts (i.e. SCN-

 was supplied as sole carbon source in the presence of external nitrogen. This could be attributed to the higher amount of available nitrogen compared to carbon (C/N ratio 0.5). Obviously the culture would find it more difficult to obtain sufficient amount of energy

is used by the consortium as nitrogen source in the presence of external

degradation utilising it as the sole nitrogen source (Bipinraj

concentration in absence of consortium (●); Cessation of bacterial

as the

degra‐

4.5

5.5

6.5

7.5

8.5

**Log (No. of bacterail cells)** 

9.5

10.5

11.5

12.5

and ammonium

degra‐

Factors influencing SCN biodegradation was restricted to the bacterial consortium isolated from activated sludge because of its high degradation efficiency compared to the consorti‐ um isolated from garden soil as mentioned earlier. Table 2 shows that degradation of SCNwas significantly influenced by the various factors tested. Optimum pH and temperature for maximum SCN biodegradation (>99.9%) was found to be 7.0 and 30°C, respectively. Under the optimized conditions of pH and temperature, the initial cell density had a substantial in‐ fluence on the biodegradation efficiency of SCN- . With initial cell density of 108 cells/ml, SCN degradation process completed within 24 h with >99.9% efficiency. As regard to car‐ bon source, the consortium culture exhibited maximum biodegradation efficiency only above glucose concentration of 5 mM.

In general, the SCN containing effluents released from various industries have pH in neu‐ tral to alkaline range. Our study showed that growth of SCN degrading bacterial consorti‐ um occurred in wide range of pH (6.0-9.0), while the optimum being 7.0. From practical applicability point of view very little or no pH adjustment would be required for the efflu‐ ents containing SCN- . Experiments also showed the unchanged pH of the solution after SCN biodegradation. This may be perhaps due to the formation of ammonia as one of the by-products of SCN- degradation, which neutralized the accumulated carboxylic acids in the medium. These results corroborate with the studies carried out by other researchers on the


ford *et al.* and Wood *et al.* glucose was supplemented at the concentration of 10 and 25 mM

(Stratford *et al.*, 1994; Wood *et al*., 1998). However, these reports did not mention optimisa‐ tion of this parameter, which needs to be worked out for economizing the process. In anoth‐ er study carried out by Patil (1999) on the biodegradation of various metal-cyanides (copper-, nickel-, zinc- and silver-cyanide), glucose was required in the range of 1-5 mM (≈ COD 100 – 500 mg/l) (Patil, 1999). Scanty information is available on the biochemical path‐

enzyme; while the sulphur moiety gets hydrolysed to sulphide, which further gets oxidised to tetrathionates via formation of thiosulphate (Stratford *et al*., 1994). It might be possible

 **by bacterial consortium at high cell density**

It can be seen from Table 3 that the bacterial consortium had low biosorption efficiency (~7-14%) at the pH values tested (6.5 to 7.5). In fact it is possible that biosorbed SCN-

could subsequently be biodegraded by the live culture used in the biodegradation process.

taking place during detoxification of SCN- by the consortium culture isolated from activated

**Initial Final**

6.5 53.29 48.22 9.51 7.0 (optimum pH) 50.94 45.97 13.68 7.5 51.03 47.50 6.91

biodegradation. For heterotrophic bacterium, Stratford *et al.* has pro‐

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

 **concentration (mg/l) % Sorption**

 **biodegradation**

dustrial effluents. Therefore, the influence of some of the commonly occurring cations such as copper, cadmium, iron, lead, nickel, zinc and anions such as sulfates, chlorides and cya‐

Table 4 shows the effect of various cations such as copper, nickel, zinc, cadmium, iron

degradation of thiocyanate was not affected in the presence of copper, nickel and zinc (degradation >90%). In presence of lead and cadmium the biodegradation efficiency was

various metal cations and anions are normally present in the various in‐

in thiocyanate-metal system. It can be seen that bio‐

to carbon dioxide and ammonia via cyanate by an inducible

, respectively

43

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

removal or toler‐

was the predominant reaction

also

for the degradation of 3 mM (≈ 174 mg/l) and 2.5 mM (≈ 145 mg/l) of SCN-

that bacterial cultures isolated in the present study also have similar SCN-

These observations confirmed that biodegradation of SCN-

by consortium culture

biodegradation was checked.

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

and lead on biodegradation of SCN-

way involved in SCN-

**3.5. Biosorption of SCN-**

**pH SCN-**

ant mechanism.

sludge.

\*Optimum pH

**Table 3.** Biosorption of SCN-

Apart from SCN-

nide on SCN-

posed the conversion of SCN-

\* Control indicates flask without culture; All the values are the average of two readings

**Table 2.** Degradation of SCN by a bacterial consortium isolated from activated sludge as a function of pH, temperature, initial cell density and glucose

biodegradation/biodetoxification free cyanide (Babu *et al*., 1993) and metal-cyanides (Patil and Paknikar, 2000b). The bacterial consortium used in the study was neutrophilic and mes‐ ophilic. Optimum temperature for thiocyanate degradation by bacterial consortium was 30°C. This is very important from the view point of actual applicability of the bioremedia‐ tion process in a tropical country like India having ambient temperature ranging from 20-40°C. Results on the impact of inoculum size clearly showed that SCN- degradation in‐ creased with the increase in inoculum size. Therefore, from the point of view of process de‐ velopment, it is essential to use a reactor capable of retaining high microbial biomass that will hasten the degradation of SCN- . Results on the influence of glucose requirement for SCNdegradation could possibly be explained on the basis of nutrient availability. Even though the available nitrogen in the form of SCN was ample the externally supplied glucose at concentrations < 5 mM limited the biodegradation process. However, at adequate glucose concentration of ≥ 5 mM complete degradation could take place. These findings confirm the utilisation of SCN as the sole source of nitrogen. In the previous studies carried out by Strat‐

ford *et al.* and Wood *et al.* glucose was supplemented at the concentration of 10 and 25 mM for the degradation of 3 mM (≈ 174 mg/l) and 2.5 mM (≈ 145 mg/l) of SCN- , respectively (Stratford *et al.*, 1994; Wood *et al*., 1998). However, these reports did not mention optimisa‐ tion of this parameter, which needs to be worked out for economizing the process. In anoth‐ er study carried out by Patil (1999) on the biodegradation of various metal-cyanides (copper-, nickel-, zinc- and silver-cyanide), glucose was required in the range of 1-5 mM (≈ COD 100 – 500 mg/l) (Patil, 1999). Scanty information is available on the biochemical path‐ way involved in SCN biodegradation. For heterotrophic bacterium, Stratford *et al.* has pro‐ posed the conversion of SCN to carbon dioxide and ammonia via cyanate by an inducible enzyme; while the sulphur moiety gets hydrolysed to sulphide, which further gets oxidised to tetrathionates via formation of thiosulphate (Stratford *et al*., 1994). It might be possible that bacterial cultures isolated in the present study also have similar SCN removal or toler‐ ant mechanism.

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

It can be seen from Table 3 that the bacterial consortium had low biosorption efficiency (~7-14%) at the pH values tested (6.5 to 7.5). In fact it is possible that biosorbed SCN also could subsequently be biodegraded by the live culture used in the biodegradation process. These observations confirmed that biodegradation of SCN was the predominant reaction taking place during detoxification of SCN- by the consortium culture isolated from activated sludge.


**Table 3.** Biosorption of SCN by consortium culture

biodegradation/biodetoxification free cyanide (Babu *et al*., 1993) and metal-cyanides (Patil and Paknikar, 2000b). The bacterial consortium used in the study was neutrophilic and mes‐ ophilic. Optimum temperature for thiocyanate degradation by bacterial consortium was 30°C. This is very important from the view point of actual applicability of the bioremedia‐ tion process in a tropical country like India having ambient temperature ranging from 20-40°C. Results on the impact of inoculum size clearly showed that SCN- degradation in‐ creased with the increase in inoculum size. Therefore, from the point of view of process de‐ velopment, it is essential to use a reactor capable of retaining high microbial biomass that

by a bacterial consortium isolated from activated sludge as a function of pH,

degradation could possibly be explained on the basis of nutrient availability. Even

as the sole source of nitrogen. In the previous studies carried out by Strat‐

at concentrations < 5 mM limited the biodegradation process. However, at adequate glucose concentration of ≥ 5 mM complete degradation could take place. These findings confirm the

. Results on the influence of glucose requirement for

**Parameter Range selected% SCN-**

Control 0

(cells/ml)

5 35.3 105 19.5 5.5 43.0 106 47.1 6 75.3 107 62.8 6.5 84.6 108 >99.9 7 >99.9 109 >99.9

8 87.6 Glucose (mM) Control 0 8.5 84.5 0.1 6.2 9 84.2 1 24.7 9.5 56.9 5 >99.9

10 16.3 20 >99.9

Temperature Control 0 10 >99.9

\* Control indicates flask without culture; All the values are the average of two readings

**biodegradation**

was ample the externally supplied glucose

will hasten the degradation of SCN-

**Parameter Range selected% SCN-**

42 Applied Bioremediation - Active and Passive Approaches

**biodegradation**

pH Control\* 0 Cell Density

7.5 89.2

20 51.8 30 >99.9 37 36.3 45 0

though the available nitrogen in the form of SCN-

SCN-

utilisation of SCN-

**Table 2.** Degradation of SCN-

temperature, initial cell density and glucose

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

Apart from SCN various metal cations and anions are normally present in the various in‐ dustrial effluents. Therefore, the influence of some of the commonly occurring cations such as copper, cadmium, iron, lead, nickel, zinc and anions such as sulfates, chlorides and cya‐ nide on SCN biodegradation was checked.

Table 4 shows the effect of various cations such as copper, nickel, zinc, cadmium, iron and lead on biodegradation of SCN in thiocyanate-metal system. It can be seen that bio‐ degradation of thiocyanate was not affected in the presence of copper, nickel and zinc (degradation >90%). In presence of lead and cadmium the biodegradation efficiency was reduced by approximately 30 and 45%, respectively. Chromium and iron significantly af‐ fected the degradation of SCN by >80%. Anions such as sulfates and chlorides (1000 μM and 10000 μM, respectively), and cyanide (0.1-1 mM) however, did not had much impact on SCN degradation.

tion. COD removal was more than 80%. It was also observed that the level of soluble copper, zinc, silver and nickel was reduced to less than 5 mg/l. There was no significant change in

**Parameter Simulated Industrial Effluent concentration (mg/l) % Removal**

The microbial process for degradation of thiocyanate was found to be highly effective in the detoxification of simulated industrial effluents. The levels of total thiocyanate, COD and metals could be brought down below the statutory limits as per Indian Standards (IS: 2490-1981). The treatment of effluent required more time as compared to the synthetic sol‐ utions. It is known that the applicability of any such process to real effluents is always complicated by the fact that effluents contain a variety of other contaminants which might interfere with or prolong the detoxification process. However, it must be emphasized that the microbial process described was highly efficient, safe and environment-friendly. In ad‐ dition, the process had the potential of becoming economically attractive if scaled-up to a sufficient level, especially as a continuous operation. Therefore, it was decided to further

 **waste in a Continuous Treatment System (CTS)**

mg/l for over a period of 30 days. The HRT of CTS was constant during the treatment period around 20 h. A closer monitoring of the CTS revealed that further reduction in the HRT was

level in the treated effluent was consistently below 0.1

Color Colourless Colourless Colourless - Turbidity Clear clear Clear pH 7.3 7.7 7.4 - Thiocyanate 51.5 < 0.1 52.4 >99 Copper 12.5 1.92 13.1 84.6 Nickel 8.1 0.55 8.0 93.2 Zinc 18.3 2.17 16.9 88.1 Iron 3.9 0.1 4.1 97.4 Sulfates 55 57 62 - Chlorides 42 45 50 - Cyanide 5.2 0.13 4.7 97.5 COD < 550 97 600 82.3

**After biodegradation Uninoculated control**

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

**efficiency**

45

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

the pH after biodegradation.

**Before biodegradation**

All the values given in the table are in mg/l, except pH

**3.8. Treatment of SCN-**

Studies in CTS showed that SCN-

**Table 5.** Composition and biodegradation of simulated industrial effluent

develop the process in continuous mode and evaluate its performance.


**Table 4.** Effect of cations and anions onμSCN biodegradation

Biodegradation of the SCN was adversely affected in the presence of metals such as iron and chromium. In case of free cyanide, it is known that cyanide ion has a great tendency to act as a ligand and can thus be found associated with metal-complexes (Pohlandt *et al*., 1983). Cyanide complexes with different metals have widely varying stabilities depending on metal oxidation states (Cotton and Wilkinson, 1972), but can be broadly classified as weakly complexed cyanides and strongly complexed cyanides. The former group includes complexes with copper, cadmium, lead, nickel and zinc (Chapman, 1992) while the latter group consists of very stable hexacyanoferrates and chromium-cyanide (Lordi *et al*., 1980). Since the chemistry of free cyanide and SCN almost being similar, the heavy metals are ca‐ pable of forming ligands with thiocyanate to form metal-thiocyanate complexes. The high stability of iron-thiocyanate and chromium- SCN might be the reason for poor degradation efficiency observed in presence of these ions.

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

The SCN- containing effluent simulated in laboratory could be effectively treated by the bac‐ terial consortium with a degradation efficiency exceeding >99.9%. The time incurred for the complete biodegradation of SCN from waste waters was less than 24 h. Table 5 shows the parameters such as pH, total cyanide, COD and metal content before and after biodegrada‐ tion. COD removal was more than 80%. It was also observed that the level of soluble copper, zinc, silver and nickel was reduced to less than 5 mg/l. There was no significant change in the pH after biodegradation.


All the values given in the table are in mg/l, except pH

reduced by approximately 30 and 45%, respectively. Chromium and iron significantly af‐

and 10000 μM, respectively), and cyanide (0.1-1 mM) however, did not had much impact

Thiocyanate (Control without culture) 0 Thiocyanate (Control with culture) >99 Thiocyanate + Copper 95.2 Thiocyanate + Nickel >99 Thiocyanate + Zinc 90.4 Thiocyanate + Cadmium 56.2 Thiocyanate + Iron 17.9 Thiocyanate + Lead 69.5 Thiocyanate + Chromate 11.8 Thiocyanate + Sulfate 74.0 Thiocyanate + Chlorides 90 Thiocyanate + Cyanide >98

biodegradation

and chromium. In case of free cyanide, it is known that cyanide ion has a great tendency to act as a ligand and can thus be found associated with metal-complexes (Pohlandt *et al*., 1983). Cyanide complexes with different metals have widely varying stabilities depending on metal oxidation states (Cotton and Wilkinson, 1972), but can be broadly classified as weakly complexed cyanides and strongly complexed cyanides. The former group includes complexes with copper, cadmium, lead, nickel and zinc (Chapman, 1992) while the latter group consists of very stable hexacyanoferrates and chromium-cyanide (Lordi *et al*., 1980).

pable of forming ligands with thiocyanate to form metal-thiocyanate complexes. The high

The SCN- containing effluent simulated in laboratory could be effectively treated by the bac‐ terial consortium with a degradation efficiency exceeding >99.9%. The time incurred for the

parameters such as pH, total cyanide, COD and metal content before and after biodegrada‐

 **from industrial effluent by bacterial consortium**

**Thiocyanate + cations/anions % Thiocyanate biodegradation**

by >80%. Anions such as sulfates and chlorides (1000 μM

was adversely affected in the presence of metals such as iron

almost being similar, the heavy metals are ca‐

from waste waters was less than 24 h. Table 5 shows the

might be the reason for poor degradation

fected the degradation of SCN-

44 Applied Bioremediation - Active and Passive Approaches

degradation.

**Table 4.** Effect of cations and anions onμSCN-

Since the chemistry of free cyanide and SCN-

efficiency observed in presence of these ions.

stability of iron-thiocyanate and chromium- SCN-

Biodegradation of the SCN-

**3.7. Degradation of SCN-**

complete biodegradation of SCN-

on SCN-

**Table 5.** Composition and biodegradation of simulated industrial effluent

The microbial process for degradation of thiocyanate was found to be highly effective in the detoxification of simulated industrial effluents. The levels of total thiocyanate, COD and metals could be brought down below the statutory limits as per Indian Standards (IS: 2490-1981). The treatment of effluent required more time as compared to the synthetic sol‐ utions. It is known that the applicability of any such process to real effluents is always complicated by the fact that effluents contain a variety of other contaminants which might interfere with or prolong the detoxification process. However, it must be emphasized that the microbial process described was highly efficient, safe and environment-friendly. In ad‐ dition, the process had the potential of becoming economically attractive if scaled-up to a sufficient level, especially as a continuous operation. Therefore, it was decided to further develop the process in continuous mode and evaluate its performance.

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

Studies in CTS showed that SCN level in the treated effluent was consistently below 0.1 mg/l for over a period of 30 days. The HRT of CTS was constant during the treatment period around 20 h. A closer monitoring of the CTS revealed that further reduction in the HRT was not possible because the bacterial cells could not be retained in the system. The COD remov‐ al efficiency after treatment was >75% for thiocyanate effluent (Table 6).

**Author details**

Yogesh B. Patil

**References**

London.

ens, pp. 491.

*gy* 85(4): 1167-1174.

Address all correspondence to: head\_respub@siu.edu.in

(SIU), Lavale, Pune, Maharashtra, India

Symbiosis Institute of Research and Innovation (SIRI), Symbiosis International University

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

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

47

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*national Journal of Chemical Sciences* 9: 1063-1072.


All the figures in the table are expressed in mg/l, except pH; \*HRT of the system was ~20 h. Figures represent average values of 30 readings taken each at 24 h interval

**Table 6.** Treatment of metal-cyanide waste waters in CTS

The results of CTS showed that SCN was degraded efficiently by the bacterial consortium with the minimum hydraulic retention time (HRT) of approximately 20 h. However, there was no reduction in the HRT of CTS further. The main reason for this was the continuous loss of active biomass from the reactor, which makes it unattractive from process economics point of view. This necessitates the immobilization of the bacterial consortium in the reactor. In principle, it is possible to retain active biomass in CTS if the culture used has an inherent property of producing wall growth. Further, biomass retention is also possible by changing the reactor design, introducing inert support material or changing nutrient supplementa‐ tion, etc. However, the consortium culture used in the present studies did not show wall growth. Also, in our studies during optimization of process parameters it was conclusively proved that degradation efficiency increased with the increase in cell number, which in turn hastens the degradation of SCN- . Thus, the above results emphasize the fact that the biore‐ mediation process developed during the course of present work is highly efficient and com‐ pletely safe. After further scale-up the bacterial process developed could have the following advantages: (i) no sludge generation; (ii) no expensive chemical additives required; (iii) very little or no pH adjustment required; (iv) the process would be easy to operate and maintain. Thus, the bacterial process developed could have the potential of becoming an economical and reliable alternative to the conventional processes employed for the treatment of SCNbearing industrial effluents on a commercial scale.

#### **Acknowledgements**

The author gratefully acknowledges the research grant provided by University Grants Com‐ mission (UGC), WRO, Pune.
