Early enrichment culture indicated that the rate of alachlor degradation was not significant (20%) due to the toxic effect of alachlor on bacterial growth or the bacteria had not been able to use alachlor as a carbon source. Data regarding alachlor biodegradation showed that one successful enrichment culture at pH=7.0 was obtained with more than 60 percent of alachlor reduction. Since control media which were not inoculated and contained sodium azide as bacterial growth inhibitor, its reduction was less than 5%, it can be concluded that its reduction was not related to evaporation, photolysis and adsorption. Therefore, the decrease of alachlor concentration corresponded solely by the bacterial consortium. Generally, low alachlor level in the enrichment culture might reflect higher capability of alachlor degradation by bacterial consortium. Plating of bacteria on solid medium revealed the presence of many different colony types, suggested the combined metabolic activities of more than one bacterium. There is a more than a decade that alachlor and atrazine are both simultaneously used for the control of broad-leaf weed in Kavar corn fields and a study was done by Dehghani et al. also showed that atrazine biodegradation was enhanced in Kavar corn field soil compare to the other soils that had not been exposed to the herbicide . The high degree of alachlor biodegradation appeared to be related to the site that had a long history of alachlor application due to the preexistence of microorganisms in the soil [21, 24]. Many researchers found that the rapid alachlor biodegradation and mineralization was only observed when the microorganisms enriched by repeated subculturing or in soils inoculated with these adopted organisms . Therefore, several application of alachlor on soil resulted in an enhancement of alachlor degradation.
Carbon sources of glucose and sodium citrate had the highest alachlor reduction rate. According to data, it is clear that sodium citrate had the major role in alachlor degradation. However, glucose as the only carbon source had resulted lower alachlor degradation compare to sodium citrate. According to data in this research, alachlor reduction rate with no carbon sources was only 26%. In laboratory conditions the carbon sources were added to support the higher growth of bacteria. Therefore, addition of carbon sources enhanced alachlor biodegradation due to cometabolism rather than direct metabolism. Past studies showed that the presence of additional substrates could initiate cometabolism of the desired compounds . Bacteria and fungi can degrade alachlor through cometabolism [20, 23, 30].
According to data shown in Figures 2 and 3, cells grown on exogenous nitrogen source have increased alachlor reduction rate significantly. Alachlor utilization is activated under nitrogen sufficient condition and reduced under nitrogen limitation. Alachlor reduction rate increased more rapidly by the addition of ammonium nitrate compare to urea. Therefore, alachlor catabolism is significantly strengthened when additional nitrogen source is available. Many investigations showed a positive effect of nitrogen amendment on alachlor biodegradation by indigenous populations in soils [31, 32]. Katz and his coworkers (2001) showed that Pseudomonas sp. strain ADP metabolized alachlor rapidly when cell grown on ammonium, nitrate, or urea . Many researchers noted using mineral nitrogen greatly increased mineralization of alachlor in soil . Organic nitrogen supplied in dairy manure increased alachlor mineralization. It can be concluded that the effect of nitrogen addition varies with the form and amount of added nitrogen.
Data regarding the effect of pH shows that as pH increased from 5.0 to 7.5, the rate of alachlor biodegradation increased. However, as pH increased from 7.5 to 8.5 caused a reduction rate in alachlor biodegradation. Based on the data obtained in the present study, pH of 7.5 is optimal for alachlor biodegradation. The reduction rate was more than 90% in this case. Dehghani et al. showed that the optimal pH for atrazine herbicide biodegradation was pH=7.0 .
Alachlor reduction rates were significantly enhanced in the inoculated soils as compared to uninoculated control soils. After 30 days, the percent of alachlor reduction was only 17.67% for uninoculated soils at initial alachlor concentration of 6.70 mg/g soil and the soil moisture of 25%. However, soil that was inoculated every 5 days with the bacterial consortium had reduced alachlor up to 74% at the same initial alachlor concentration and the soil moisture by Day 30 (Table 1). As initial alachlor concentration increased from 6.7 to 53.3 mg/g soil, alachlor reduction decreased from 74 to 33% (inoculated soils and soil moisture 25%). The decrease in alachlor reduction at 53.3 μg/g soil was possibly due to the result of complex interaction between microbial activity and nutrient availability. Therefore, unbalanced nutrient supply was probably responsible for the decrease. According to Table 1, enhanced alachlor reduction rates occurred with an increase in soil moisture from 7 to 25%. The percent of alachlor reduction increased from 35.83 to 74% as the soil moisture increased from 7 to 25% (at initial alachlor concentration of 6.7 mg/g soil). Many researchers found that higher herbicide reduction rate occurred with an increase in soil moisture. Soil moisture influences microbial processes through direct effect (e.g. water availability) or indirect effects such as solute diffusion, chemical availability and aeration . Therefore the positive effect of increasing soil moisture was probably due to increased microbial mobility, solute diffusion and chemical availability which all had an indirect effect on alachlor degradation. The alachlor degradation reached a plateau at about 2 weeks. There are many reasons that degradation of alachlor became constant. One of the possibilities for the plateau is the shortage of necessary nutrients for growth of microbe and the other reason might be related to the nature of the alachlor. The bioavailability of a compound decreased with time until a minimum value is reached; at that point, no more degradation of alachlor will occur. Once the microorganisms degrade the available substrate present in the soil, the degradation rate is limited to the rate of desorption of the sorbed fraction back into the soil solution [35, 36]. In this study, more than 70% of alachlor was degraded in 30 days of incubation periods (Figure 5). Assuming a pseudo-first order reaction for the disappearance of alachlor, a plot of the natural logarithm of initialized alachlor concentration (C/C0) versus time resulted in a rate constant equal to 0.0968 1/d (Figure 6). Alachlor degradation exhibited a short half-life of approximately 7.16 d. The initial slow degradation rate of alachlor was followed by a much faster degradation rate that lasted about Day 5 and then the degradation rate became slower and finally remained constant until the end of incubation period. Another study demonstrated that the half-life for alachlor was only 3.0 days. While the measured half-life in our study is about 2.5 times more than the Chirnside et al. study .