The isolated Bacillus cereus Ct3 and Pseudomonas aeruginosa WW7 was resistant up to 400mgL-1, whereas Klebsiella oxytoca Ct27 showed resistance only up to 250mgL- 1
. With the initial concentration of 300mgL-1, Bacillus cereus Ct3 degraded 87% of CP in 7 days. Similarly, Pseudomonas aeruginosa WW7 was able to degrade 79% CP with initial concentration of 300mgL-1. Concentration above 300mgL-1 showed longer lag phase. These longer lag phases might because of the requirement of larger microbial number and acclimation period to begin enhanced biodegradation (Karpouzas and Walker 2000). The order of degradation ability was as follows;
Bacillus sp. > Pseudomonas sp. > Klebsiella sp.
As the degradation process starts the amount of pesticide residue start decreasing and the amount of metabolites starts increasing (Boettcher et al. 1992). At any time during biodegradation, rate of biodegradation is proportional to the residuals concentration of pesticide. CP biodegradation involves phopshatase and phospho-mono-esterase (enzymes) for the hydrolyzation of O-P bonds (Briceño et al. 2012). This releases
phosphorous atom as phosphorous source by the microbes (Singh et al. 2004; Singh and Walker 2006). The rate and limit of CP degradation is different by different microbes. Xu et al. (2007) reported Serratia sp. and Trichosporon sp. capable of degrading 100mgL-1 of CP in 24h. Enterobacter strain B-14 was reported for biodegradation of 250mgL-1, with longer lag phases at higher concentration (Singh et al. 2004). However, maximum degradation (up to 89%) of 1000mgL-1 has been reported by B. pumilus C2A1 (Anwar et al. 2009). Instead of single bacterium specie, consortium of bacteria can be used with even higher degradation rate and limits. For instance, consortium of P. putida, P. stutzeri and Klebsiella sp. was able to degrade 500mgL-1 of CP (Sasikala et al. 2012). This variation in degradation potential at different concentration is due to the presence and absence of genes (mpd) and enzymes (phopshatase and phospho-mono-esterase) involved in CP degradation (Lu et al. 2013).
All the 3 isolates (Bacillus cereus Ct3, Pseudomonas aeruginosa WW7 and Klebsiella
oxytoca Ct27) showed different range of temperature tolerance, which was in the range of 25-35oC, 25-30oC and 30-35oC, respectively. Optimum temperature exhibited by Bacillus sp., Pseudomonas sp. and Klebsiella sp. was 30oC, 30oC and 35oC, respectively. The results of other studies were in the same alignment (Abboud et al. 2007; Anwar et al. 2009). Sarkar et al. (2010) was able to isolate Pseudomonas sp. from tea rhizosphere, which showed 69% degradation of propargite (insecticide) at 30-35oC. Similarly Enterobacter sp. show optimum temperature of 35oC. Degradation ability of Enterobacter sp. did not effected significantly within the range of 25-40oC the, but further decrease (below 15oC) or increase (above 50oC) in temperature drastically reduced the biodegradation (Singh et al. 2006). 37oC has been recommended as optimum temperature for Agrobacterium, Bacillus cereus and
Pseudomonas aeruginosa (Maya et al. 2011). Similarly, biodegradation study by Anwar et al. (2009) reported 90% CP degradation (300mgL-1) by Bacillus sp. at 37oC. Abboud et al. (2007) reported 90% degradation of alkylbenzosulfonate and sodium dodecyl sulfate by Acinetobactor calcoaceticus and Pantoea agglomerans at 30oC.
Verticillium sp. (a fungal strain) capable of degrading CP, showed maximum efficiency at 35oC, which was 1.12 times faster than 20oC. Liu et al. (2012) experimentation revealed that Bacillus cereus worked maximum at 30oC and showed 78% degradation. Whereas, at 25oC the degradation was 75%. Likewise, Li et al.
(2008) revealed 30oC optimum temperature with 98% CP degradation by
Stenotrophomonas sp., Pseudomonas sp. and Bacillus sp. Change in temperature influences the solubility, bioavailability and other physio-chemical properties. This may be one of the reasons of different biodegradation behavior at different temperature (Mohan and Reddy 2013). Another possible justification of variation in degradation at different temperature is the optimum temperature of enzymes, which are involved (Baczynski et al. 2010). This wide temperature bearing ability favors the isolates to work under changing environmental conditions (Liu et al. 2012).
Experimentations on pH change revealed that Bacillus sp. is very spacious toward pH tolerance (7-8.5). However, maximum degradation was observed at pH 8.5, just by increasing pH from 7 to 8.5, 11% increase in degradation occurred. Similar trend in pH tolerance was shown by Pseudomonas sp. (7-8.5). It showed 10.2% increase by changing pH from 7 to 8.5. However, tolerance range of Klebsiella was in a narrow range. It efficiently degraded only in the range of 7-8. Beyond this pH range the degradation efficiency decrease drastically, degradation at 7 and 8 pH differ about 15.7%. Possibly, chlorpyrifos degrading enzymes have optimum activity at high pH (Swetha and Phale 2005). Results of the other studies also conform this trend (Xie at al. 2010; Abboud et al. 2007). This wide pH range sounds very significant. As the environmental conditions keeps on changing and hence microbes with wide tolerance range have better chance of survival. Singh (2008) reported rapid chlorpyrifos degradation by an Enterobacter sp at higher pH, while it was significantly slow at low pH. Conversely, Karpouzas and Walker (2000) reported Pseudomonas putida (epI and epII) which quickly degraded organophosphate pesticide (ethoprophos) from pH 7.6 to 5.5. Bacillus cereus demonstrated optimum pH of 7, but pH 6 and 8 also show significant similar result compared to pH 7. However pH greater than 8 and less than 6 significantly inhibit CP degradation (Liu et al. 2012). Beside bacteria, fungal strains can also be used for biodegradation of CP and ecological restoration. Fang et al. (2008) isolated Verticillium sp. from contaminated soils. This fungal strain was more efficient at pH 7. At pH 5 and 9 it exhibited 1.12 and 1.04 times slower degradation rate. Optimum pH of 8.5 was also reported by Abboud et al. (2007), who reported 90% degradation of alkylbenzosulfonate and sodium dodecyl sulfate by Acinetobactor
calcoaceticus and Pantoea agglomerans. Xie et al. (2010) extracted free enzymes capable of degrading CP from WZ-I (Fusarium fungus). These enzymes under
immobilized conditions showed optimum pH of 8 for CP degradation. Whereas, optimum pH for free enzyme was 6.8. This difference in optimum pH of free and immobilized enzyme was due to sodium alginate as embedding medium (Xie et al. 2005).
Addition of carbon sources stimulates microbes to degradation more efficiently (Anwar et al. 2009). In present results, the glucose significantly elevated biodegradation rate. The difference in CP degradation with or without glucose was 34%, 39% and 19% by Bacillus sp., Pseudomonas sp. and Klebsiella sp., respectively. This enhancement in degradation is because of increase in initial growth of microbial strains, which leads to the more number of microbes. The order of effectiveness of tested carbon sources is as follows;
Glucose > yeast extract > starch > no added supplement
Thus more number resulted in more degradation (Swetha and Phale 2005). This boosts in biodegradation also represents the phenomena of co-metabolism, where addition of easily metabolized organic matter boost degradation of such compounds which are usually not used as energy and carbon source (Qiu et al. 2007). Earlier findings suggested the use of glucose as co-substrate and the process of co- metabolism is widely accepted for biodegradation management (Sarkar et al. 2010). Pino and Peñuela (2011) reported increased degradation of CP up to 100% within 5 days of incubation with the addition of glucose by a consortium (Pseudomonas
Putida, Pseudomonas aeruginosa, Klebsiella sp, Flavobacterium sp and
Acinetobacter sp). Anwar et al. (2009) isolated Bacillus pumilus from CP contaminated agri-soil. This strain completely mineralized 50mgL -1 of CP in just 3 days in presence of glucose. Other carbon sources like yeast extract and nutrient broth also showed positive increase in degradation up to 86% and 92%. However, addition of carbon source may not always favors biodegradation. According to Singh et al. (2004), glucose presence show negative effect on Enterobacter strain and reduced degradation rate. But after 3 days the same strain started consuming CP again. This difference might be because of preference of microbe over different carbon source. Successfully used carbon sources include, glucose, glalactose, maltose, starch, carboxyl methyl cellulose, xylose, mannose, sorbitol and salicin. Among all these carbon sources glucose showed most significant enhancement and next to that was
glalactose. Starch and maltose did not show any significant effect (Sarkar et al. 2010). Maltose has the tendency to enhance degradation of alkylbenzosulfonate and sodium dodecyl sulfate at concentration of 0.2% by Acinetobactor calcoaceticus and Pantoea
agglomerans (from wastewater). However, succinate failed in enhancing significant degradation (Abboud et al. 2007).
The results of the present study reveals that the inoculum density of 105 (CFUml-1) is sufficient for excellent biodegradation. Increase in degradation rate can be achieved by increasing the inoculum size. However, small inoculum size (below 105 CFUml-1) leads to the poor degradation with longer lag phase. Chen and Alexander (1989) suggested that this longer lag phase represents the time for growth of active bacterial population which is in small number. And rapid degradation starts when their number reach to a certain sufficient level. Before that significant number of active bacterial population, biodegradation cannot proceed (Anwar et al. 2009). This significant number depends on resistant level and on the chemical nature of pesticides to be degraded (Fang et al. 2008). Other studies also support present results. Li et al. (2008), observed 98% CP degradation with 108 cellsml-1. Singh et al. (2006) reported rapid CP (250mgL-1) degradation with initial inoculum densities above 105CFUml -1. In the same study, 103 and 104 CFUml-1 showed longer lag phases and inoculum densities below 103 did not degraded CP significantly. Whereas, Anwar et al. (2009) reported that inoculum density below 105CFUml-1 show longer lag phase and rapid degradation was observed at 109 CFUml-1 in 5 days of incubation. Different researchers have used different initial inoculum densities for CP biodegradation, like 2 x 108 cellsg-1 for 50mgL -1 (Lakshmi et al. 2008), 108 cellsml-1 for 100mgL -1 (Li et al. 2008), 108 CFUml-1 for 200mgL-1 (Maya et al. 2011), 107 CFUml-1 for 1500mgL-1 (Pino and Peñuela. 2011) and 106 cellsml-1 for 100mlL-1 (Liu et al. 2012). Similarly, Korade and Fulekar (2009) reported 106 CFUml-1 of Pseudomonas nitroreducens and 2% of 1 x 106 CFUml-1 for 50mgL-1 of cypermethrin (Chen et al. 2012).