Statistical Enhancement of Cyanide Degradation Using Microbial Consortium

Cyanide containing wastewater is a potent health hazard for humans and components of ecosystems [1].The toxicity of cyanide is quite high due to its ability to poison the respiratory system by inhibiting the final transport of electrons from cytochrome C oxidase to oxygen, preventing production of ATP. The cyanide containing wastewater is produced by various industries like acrylic fibers and resin production industries, coal industries, cassava starch production industries, plastic production and mining industries etc. [1,2].


Introduction
Cyanide containing wastewater is a potent health hazard for humans and components of ecosystems [1].The toxicity of cyanide is quite high due to its ability to poison the respiratory system by inhibiting the final transport of electrons from cytochrome C oxidase to oxygen, preventing production of ATP. The cyanide containing wastewater is produced by various industries like acrylic fibers and resin production industries, coal industries, cassava starch production industries, plastic production and mining industries etc. [1,2].
The protection of the environment and safety of ecosystems can be maintained by treating wastewater before being discharged into water bodies [3]. Various physical, chemical and biological methods are being used for this purpose. Chemical methods include alkaline chlorination, ozonisation, and wet-air oxidation [4]. High operational cost vis-à-vis generations of chemical wastes are limitations of these processes [5]. However, microbial detoxification of cyanide has emerged as an inexpensive, ecofriendly, and viable alternative to physiochemical processes for the treatment of cyanide in industrial effluents or contaminated sites [6]. The microorganisms reported to degrade cyanide include Bacillus sp. [7], Pseudomonas flourescens [8], Fusarium solani, Trichoderma polysporum, Penicillium miczynski, Azotobacter vinelendi [9] and Serratia marcescens RL2b [10]. Among these, Azotobacter vinelendi degrades 69% of 4 mmol l -1 sodium cyanide while Bacillus sp. CN-22 has been reported to effectively degrade cyanide upto 200 mg l -1 concentration [7] and Serratia marcescens RL2b which is capable of degrading 780 mg l -1 of cyanide in a packed bed reactor completely [5]. However, microorganisms also have their own limitations in terms of growth in specific environment, substrate tolerance and degradation which is evident from previous reports. In the current study, a consortium based approach followed in order to achieve higher substrate tolerance and degradation of cyanide. Three bacterial isolates, Enterobacter sp. RL2a, Serratia marcescens RL2b, and Achromobacter sp. RL2c, which were isolated from different habitats, were identified and then cultivated together in consortium followed by optimization for in vitro cyanide degradation using response surface methodology.

Isolation of cyanide degrading bacteria
Different samples were collected from rhizosphere soil of cyanogenic plants and industrial wastewater sites of Himachal Pradesh, India. These samples were immediately processed in laboratory for the isolation of cyanide degrading bacteria. Enrichment culture technique using minimal salt medium and glycerol [1%] and potassium cyanide [780 mg l -1 ] as sole source of carbon and nitrogen respectively were used for isolation [10]. After third enrichment, samples were serially diluted and plated on minimal salt medium agar supplemented with potassium cyanide [780 mg l -1 ]. The pure colonies were obtained by streaking on nutrient agar plates. Three best isolates [RL2a, RL2b and RL2c] on the basis of their cyanide degradation potential were selected, identified using biochemical tests [11] and 16S rDNA sequencing data.

Isolation of cyanide degrading bacteria
Three cyanide degrading bacteria were selected after screening of eighty six isolates. Morphological examination of colonies of RL2a and RL2b and RL2c (Figure 1) isolates on agar plate showed them to be creamish white in color, concave, smooth and wet. The cells were large and rod like in case of RL2a (Gram negative) and RL2b (Gram negative) whereas, RL2c (Gram negative) had coccal rod like cells. The biochemical characteristics of the isolates are given in Table 1. No antagonistic activity was observed among these isolates.

Molecular characterization
The partial 16S rDNA sequences of RL2a, RL2b and RL2c were determined and phylogenetic tree was constructed ( Figure 2). Comparison of ribosomal DNA gene sequence analysis revealed a strong relationship between isolate RL2a and members of genus Enterobacter, RL2b and members of genus Serratia and RL2c and members of genus Achromobacter. The isolate RL2a had 99% sequence similarity with Enterobacter sp.; RL2b had 98% sequence similarity with Serratia marcescens strain XJU PA-6 and RL2c had 99% sequence similarity with that of Achromobacter sp. The sequences were deposited in GenBank database with accession number KF225466, JX549409 and KF225467, respectively. On the basis of physiological and biochemical characteristics and results of 16S rDNA gene sequence comparison, the selected isolates were identified as Enterobacter sp. RL2a, Serratia marcescens RL2b and Achromobacter sp. RL2c ( Figure 2).

In vitro cyanide degradation by microbial consortia
The antagonistic effect of selected bacterial isolates was studied in order to check whether the consortium components coexist with each other or not. The results showed that bacterial isolates do not interfere with each other during growth in culture medium. A very little difference in degradation rate was observed by the bacteria when different percentage of initial inoculum volume was used ( Figure 3). Thus, the initial 2% inoculum volume of cultures was used for further experiments. Near neutral pH values support degradation of cyanide as well as the growth of consortia as evident from Figure 4. Cyanide degradation was maximum at pH 6.0 and it went on decreasing with increase in pH. Time course of cyanide degradation was also reduced from 60 h to 48 h. In the present investigation, 99% degradation of cyanide was observed at 30°C with biomass of 5 mg ml -1 ( Figure 5). Maximum cyanide concentration which could be tolertated by consortium is 20 mM ( Figure 6). Technologies] and were sequenced at Xcelris Genomics, Ahmedabad, India. Related matching sequences were downloaded from the GenBank database [NCBI, USA] using blast search program [13]. The sequences were aligned using multiple sequence alignment software, Clustal W version 2.0. A phylogenetic tree was constructed with MEGA align software version 5.1 [14] based on the partial 16S rDNA sequences of bacterial species similar to isolate RL2a, RL2b and RL2c.

In vitro cyanide degradation by microbial consortium
Three selected bacterial isolates were checked for their antagonistic activity. Two bacterial isolates against each other were plated on nutrient agar plates supplemented with cyanide. Different combinations were made and plated. CFU ml -1 [Colony forming units] was also calculated by plating three isolates on nutrient agar plates. Microbial consortium was then optimized in order to achieve maximum cyanide degradation in vitro. Various parameters were taken into account, i.e., inoculum volume, pH, temperature and cyanide concentration. Different combinations viz. consortium (RL2a, RL2b and RL2c); RL2a and RL2b; RL2a and RL2c; RL2b and RL2c; RL2a; RL2b; RL2c were made and biomass (mg ml -1 ) and percent cyanide degradation was observed after different time intervals.

Optimization of reaction conditions by response surface methodology [RSM]
Plackett Burman design was used to screen the factor having positive effect on degradation of cyanide by microbial consortia. Various reaction parameters and physical parameters were screened for nine variables at two levels, minimum and maximum. Nine different variables, i.e., pH (5.0-9.0), temperature (20-

Optimization of selected variable using central composite design (CCD)
The concentration of different independent variables which showed positive effect (pH, time , strain RL2b) for cyanide degradation was optimized by using Central Composite Design (CCD). Second-order polynomial coefficient were calculated and analyzed by using Design Expert software 8.0.4.

Validation of statistical model
The statistical model was validated for the degradation of cyanide by performing experiment at shake flask under predicted set of conditions.

Assay method
Samples withdrawn in small aliquots at different intervals of time were centrifuged and the degradation of cyanide in the supernatant was assayed using picric acid assay method [15]. Absorbance was measured at 520 nm and concentration of cyanide was calculated from standard curve of cyanide estimation (prepared by assaying KCN ranging from 1.5 µmol l -1 -13.5 µmol l -1 ).

Statistical analysis
All the reactions were carried out in triplicates and error bars represent the standard deviation.

Factorial design for enhancing cyanide degradation by resting cells of microbial consortium
Screening of important parameters using plackett-burman design : Resting cells of components of microbial consortium were prepared and harvested after 36 h at 6000 x g for 10 min. These cells were then utilized to screen important parameters using Plackett-Burman design. Various parameters used in the study are listed in Table 2. Total of nine variables were used in the study. Pareto chart

Comparative study of bacterial cyanide degradation using different combinations
Complete degradation of cyanide was observed in consortium with high biomass concentration (5 mg ml -1 ) after 36 h. Bacterial isolate RL2c also showed good degradation. Combination of RL2a and RL2c also showed 80% degradation. But maximum degradation was recorded in consortium which revealed the synergetic effect of all components of consortia (Figure 7).   Lack of fit test for CCD model: ANOVA analysis of the central composite design (CCD) results was performed and four process orders were suggested by Design Expert 8.0.4. Quadratic process order proved best and used for further analysis due to low standard deviation (0.109), high R-squared value (0.847) and low press value (0.15) for further analysis. The analysis of the quadratic regression model suggested that the model was very significant as correlation coefficient (0.847) closer to 1 denotes better correlation between the observed and predicted responses (Table 3). Three-dimensional plots were generated for regression analysis of CCD design of pair wise combination of was constructed to find out the positive variables which affect cyanide degradation (Figure 8). Three variables i.e. time, resting cells of isolate RL2b and pH had shown positive effect on cyanide degradation while other factors had shown a negative effect. Subsequent experiments were performed using these three variables in central composite design.

% Cyanide degradation
Central composite design: For the determination of optimum level and combined effect of different variables (pH, time and strain RL2b), a central composite design was used, and a second order polynomial equation was derived to explain the dependence of cyanide degradation on these three variables by microbial consortium.   three factors for cyanide degradation. The effects of the independent variables and combined effects of each independent variable upon the response variable were described by plotting 3d graphs (Figure 9a-9d).

Validation of model:
The maximum cyanide degradation was achieved by performing experiment on the basis of the above statistical information. Perturbation plot (Figure 9a) showed the optimum value for variable pH 7.0, time 55 min and 125 µl cells of strain RL2c (1.5 mg ml -1 ). The model was validated by performing the experiment under optimum conditions, which resulted in 63% cyanide degradation. By performing factorial design, there was 1.3 fold (33%) increases in cyanide degradation.

Discussion
Eighty six cyanide degrading microbes were isolated from various soil and water samples. Soil samples were collected either from rhizosphere of cyanogenic plants e.g. cassava, peach, almond and apple because these places have more possibility and probability due to continuous enrichment of soil with different cyanogenic compounds released for defense purposes. Water samples were collected from electroplating, pesticide and textile industries which were using simple and complex cyanides. Three best cyanide degrading isolates were selected after screening. These isolates were then monitored for their antagonistic activity against each other which is very important property for consortium development. No antagonistic activity was found in these isolates. Three isolates were then identified by various biochemical and morphological features and 16S rDNA sequencing. These were gram negative, catalase positive and produce H 2 S ( Table  1). The phylogenetic studies of the organisms using Clustal Omega and Mega Align software were carried out ( Figure 1). Enterobacter sp. RL2a and Achromobacter sp. RL2c are being reported for the first time as cyanide degrading microorganisms. Serratia marcescens RL2b has been previously reported as an efficient cyanide degrading bacterium [10].
The volume of the initial culture, incubation temperature, pH and substrate concentration supplemented with appropriate ratio of carbon, nitrogen and other nutrients added to the medium control microbial growth and activity [16]. The authors reported that degradation efficiency was poor in media that were supplemented with Beta valgaris, Ipomoea batatas, Ananas comosus and spent brewer's yeast extracts at all the cyanide concentrations evaluated (400 mg l -1 ). The ability of the white rot fungus Trametes versicolor to degrade cyanide at a maximum cyanide concentration of 400 mg CN l -1 , where the degradation efficiency was less than 30% was reported [17]. The immobilized cells of Klebsiella oxytoca were used for the degradation cyanide at a maximum concentration of 157 mg CNl -1 where the microbe had achieved a degradation efficiency of 26% when cellulose acetate was used as a carbon source [18]. The bioaugmentation of cyanide coke waste water (14 mg l -1 ) using Cryptococcus humicolus and an unidentified cyanide degrading organism was reported [19]. At high concentration (500 mg l -1 ), only about 45% of cyanide was degraded within 4 days using bacterial isolate C2 [20]. Bacillus sp. CN-22 was isolated from a cyanide-contaminated electroplating sludge and found to be able to tolerate a CN − concentration of 700 mg l −1 [7]. Klebsiella oxytoca can remain active at a CN − concentration of 26mg l −1 [21] and the immobilized cells of K. oxytoca could tolerate a higher CN − concentration of 78 mg l −1 [18]. Azotobacter vinelandii has CN − resistance at 104 mg l −1 [22], and Serratia marcescens RL2b could tolerate 780 mg l -1 of potassium cyanide [10] which is the highest concentration of cyanide tolerated by microorganisms reported so far. In present study, minimal salt medium [23] was used, supplemented with glycerol and yeast extract for growth and 15 mM cyanide (930 mg l -1 ) in order to reduce the overall cost of operation.
Maximum cyanide degradation (99 %) was achieved with 2% initial volume of culture (consortium) in vitro, 30°C temperature and pH 6.0. The initial time (3 days) used for complete degradation of added cyanide was reduced to one and a half day (36 h) by optimizing culture conditions. Consortium degrades cyanide efficiently in acidic pH (6.0). With the increase in pH degradation rate falls. The decrease may be due to non-availability of favorable environment for the isolates at high pH. Microbial consortium is mesophilic in nature. Maximum concentration of cyanide that can be tolerated by the consortium reflects the degradation potential of the components of consortium. The bacterial isolates were used in different combinations to see whether the combination of microorganisms used for consortia worked well or some other combinations for efficient cyanide removal are required. Comparative analysis revealed that the combination used in the study worked very well for cyanide degradation. It can be depicted that an   increase in concentration of cyanide from 20 mM leads to retardation of growth ( Figure 4) which may be due to denaturing of bacterial repiratory enzymes at high cyanide that ultimately leads to less ATP production hence decrease in growth. 20 mM of cyanide was degraded completely in 36 h.
The Response Surface Methodology (RSM) was used in evaluation of optimization of biodegradation of cyanide in reactions. This method is based on analysis of response as affected by some factors and its objective is to determine the optimum condition of the response. The important factors which affect cyanide biodegradation were pH, time and strain RL2c. The analysis of variance (ANOVA) of the quadratic model demonstrates that the model was highly significant for bacterial isolates C2 [20]. Zhu et al. used RSM to determine the optimum levels of different variables in the microwave-assisted extraction of astaxanthin from Phaffia rhodozym. The cyanide content decreased with increasing temperature and pH in the range of 30 to 36°C and 9-10.5, respectively. However, the degradation rate was reduced for a further increase in temperature and pH value. The optimum conditions were at 23°C, pH 10.3 and glucose concentration 0.44 (g l -1 ) which could provide 24 ppm for predicted cyanide content and 26 ppm for experimental content [20]. Bacillus sp. CN-22, which could tolerate the highest CN − concentration of 700 mg l −1 (11 mM), under the optimal conditions of 193 rpm rotary speed, 2.38% inoculum, 31°C temperature, 10.3 initial pH, and the CNconcentration in the effluent was reduced from 200 to 6.62 mg l −1 after incubation for 72 h. A mesophilic alkali-tolerant bacterial consortium belonging to the Bacillus genus was evaluated for its ability to biodegrade high free cyanide (CN−) concentration (up to 500 mg CN l -1 ), subsequent to the oxidation of the formed ammonium and nitrates in a continuous bioreactor system solely supplemented with whey waste. The whey supported Bacillus sp. culture was not inhibited by the free cyanide concentration of up to 500 mg CN l -1 , with an overall degradation efficiency of ≥ 99% with subsequent nitrification and aerobic denitrification of the formed ammonium and nitrates over a period of 80 days [24,25]. However, the problems with above mentioned alternatives are low cyanide tolerance, high pH and low degradation rate using single microorganism. To avoid these drawbacks, microbial consortium was developed which exhibit high cyanide tolerance at near neutral pH [6-6.5] and optimum temperature (30°C). Response surface methodology studies showed that the quadratic model works well for the consortium attaining 63% degradation in single hour for 20 mM (1300 mg l -1 ) cyanide. A total of 1.3 fold increase in degradation rate was achieved. Complete degradation of 20 mM cyanide was achieved in 6 h.

Conclusion
The consortium developed in the present study could degrade 20 mM cyanide completely in simulated wastewater and tolerate 30 mM at 30°C and pH 6.0. Response surface methodology approach resulted in 1.3 fold increase in cyanide degradation. Removal of cyanide from waste water using combination of microorganisms proved to be a better approach. The consortium have shown highest rate of degradation and tolerance for cyanide among the hitherto reported microbial systems.