3.1. Determination of Kinetic Parameters
The half saturation constant (Ks) and the maximum specific substrate utilization rate (qmax) were determined by fitting Equation (2) to batch experimental data with the varying initial 1,4-dioxane concentrations using MATLAB software (The Math Works, Inc., Natick, MA, USA). The effect of cell decay was minimized by using a relatively high concentration of biomass for the experiments. The yield coefficient was estimated by measuring the total suspended solids (TSS) concentration of batch incubations containing 200 mg/L 1,4-dioxane. The values for Ks, qmax, and Y are 7.47 mg/L, 0.0051 h−1, and 0.434 mg/mg-VSS, respectively.
The biodegradation of 1,4-dioxane at varying concentrations of biomass was determined in batch experiments, and the results are shown in Figure 2
. The degradation of 1,4-dioxane is well-described by the model based on the Monod equation.
3.2. The Effects of Easily Degradable Carbon Sources
The effects of easily degradable organic materials were investigated by monitoring the biodegradation of 1,4-dioxane in the presence of dextrose with the initial biomass concentration of 500 mg-SS/L. Figure 3
shows the soluble COD change of the cultures in the presence of varying amounts of dextrose.
It appears that the biodegradation rate of 1,4-dioxane is not affected by the presence of dextrose. The initial rapid decrease of COD reflected the rapid biodegradation of dextrose. However, once the dextrose was degraded, the further decrease of COD became identical regardless of the initial dextrose concentrations. The direct measurements of 1,4-dioxane during the experiments (Figure 4
) clearly showed that the 1,4-dioxane degradation rate was not affected by the presence of dextrose, indicating no substrate competitiveness on the specific enzymes involved in 1,4-dioxane biodegradation.
Although the mixed culture was able to grow well on dextrose, it is not clear that the specific 1,4-dioxane-degrading bacteria or consortium grown on dextrose is capable of degrading 1,4-dioxane.
To determine whether the bacteria grown on glucose are capable of degrading 1,4-dioxane, the cells were recovered after repeated washing by centrifugation and re-suspended into BSM flasks containing 100 mg/L 1,4-dioxane. The biodegradation of 1,4-dioxane and cell growth were monitored, and the values are plotted in Figure 5
and Figure 6
As shown in Figure 5
, although the initial amounts of biomass from the cultures enriched in the presence of 300 mg/L of dextrose (B,C) were 630% greater than those of the others (A,D), the difference in cell mass did not make any difference in 1,4-dioxane biodegradation. However, the cultures grown on 1,4-dioxane (A,B) showed 167% faster degradation, presumably due to the increased population of the specific bacterial consortium growing on 1,4-dioxane (Figure 6
). It appears that the augmented biomass grown on dextrose did not contribute to the biodegradation of 1,4-dioxane at all. This observation is important when treating 1,4-dioxane in real wastewater because high BOD is very common in most 1,4-dioxane-containing wastewaters, including textile industry wastewaters.
3.3. The Effects of Structural Analogs
Three structural analogs including THF, 2-methyl-1,3-dioxolane, and 1,4-dioxene were tested for their effects on the biodegradation of 1,4-dioxane. THF is one of the most commonly employed cyclic ethers for industrial applications, and has been studied either as a cometabolic compound or inhibiting compound in 1,4-dioxane biodegradation [33
]. The biodegradation of both 1,4-dioxane and THF was determined. THF could be degraded completely by the mixed culture as a sole carbon source, as shown in Figure 7
. Soluble COD measurements confirmed no significant accumulation of byproducts. The estimated qmax
was 0.017 mg COD/mg VSS/h, almost twice the value (0.009) for 1,4-dioxane. Figure 8
shows the effects of THF on biodegradation of 1,4-dioxane.
THF clearly inhibited the biodegradation of 1,4-dioxane, and the inhibition period seemed to depend on the concentration of THF. Figure 8
shows that the total 1,4-dioxane removal time went from 55 h to 61, 72, or 84 h, with a range 111% to 153% longer in the presence of THF. The reflection points at which 1,4-dioxane concentration started falling in the presence of 75 and 150 mg/L of THF roughly match the points of complete THF degradation (Figure 7
). It appears that the biodegradation of 1,4-dioxane recovered only after complete degradation of THF, indicating that THF is a much more favorable substrate over 1,4-dioxane for the consortium. Additionally, 1,4-dioxane degradation was enhanced (removal rate from 1.81 to 2.12 mg/L h−1
) after 36 h, at which point the residual THF disappeared or reached relatively low levels. This finding implies that the mixed culture grown on THF is capable of actively degrading 1,4-dioxane, indicating the same enzymes are involved in the biodegradation of both compounds. Chen [44
] also claimed that enzymes produced by THF utilization could degrade 1,4-dioxane and increase the degradation rate.
A separate experiment confirmed that the mixed culture grown on THF was capable of actively degrading 1,4-dioxane, indicating the same enzymes are involved in biodegradation of both compounds. The cells grown on THF and 1,4-dioxane respectively were recovered after repeated washing by centrifugation, and re-suspended into BSM flasks containing 100 mg/L 1,4-dioxane. Figure 9
shows that every culture was able to degrade 1,4-dioxane at the same degradation rate. Thus, the inhibitory effects of THF may be explained by competitive inhibition. Zenker [33
] also showed that THF has a higher specific utilization rate than 1,4-dioxane for a mixed culture. Similar inhibition effects of THF on 1,4-dioxane were also reported in their research.
Unlike THF, studies on the effects of 2-methyl-1,3-dioxolane and 1,4-dioxene could not be found in the literature. The compounds were identified at relatively high concentrations in the textile wastewater used in this study. Figure 10
shows the chromatogram of GC/MS analysis of the raw textile wastewater. Typically, among all textile wastewaters, the concentration of 2-methyl-1,3-dioxolane is higher than 1,4-dioxane, and the concentration of 1,4-dioxene is the lowest.
The biodegradation of 1,4-dioxane in the presence of 2-methyl-1,3-dioxolane and 1,4-dioxene is shown in Figure 11
and Figure 12
As shown in Figure 11
, the biodegradation of 1,4-dioxane was inhibited in the presence of 2-methyl-1,3-dioxolane, while the biodegradation of 2-methyl-1,3-dioxolane was not affected by 1,4-dioxane. The removal time of 1,4-dioxane went from 21 to 36 h, representing a 170% increase in the removal time in the presence of extra carbon sources. As the concentration of 2-methyl-1,3-dioxolane decreased, the biodegradation rate of 1,4-dioxane recovered. Similar behaviors were observed with 1,4-dioxene, the dehydrogenation product of 1,4-dioxane. The biodegradation of 1,4-dioxene occurred faster than 2-methyl-1,3-dioxolane and the retardation of 1,4-dioxane biodegradation was relatively short. Again, 1,4-dioxene biodegradation was not affected by the presence of 1,4-dioxane. All the three co-occurring compounds appeared to have a much higher affinity over 1,4-dioxane for the enzymes acting on the rate-limiting step of 1,4-dioxane biodegradation. It may require further study to elucidate why the microbial consortium showed less affinity to 1,4-dioxane than other structural analogs such as THF, 2-methyl-1,3-dioxolane, and 1,4-dioxene. Practically, these results indicate that the biological treatment of 1,4-dioxane in some wastewaters requires additional retention time mainly due to the co-existence of various structural analogs. The biodegradation experiments with the raw wastewater showed the inhibitory effects of both 2-methyl-1,3-dioxolane and 1,4-dioxene.
As shown in Figure 12
, 1,4-dioxane biodegradation was retarded with range of 143% to 171% in the raw wastewaters, and the period of retardation was longer when the initial 2-methyl-1,3-dioxolane concentration was high with the same biomass condition (Figure 13
On the other hand, Zenker [33
] claimed that the presence of THF may be beneficial to 1,4-dioxane treatment by both stimulating the initial growth of 1,4-dioxane-degrading bacteria and maintaining their presence in a bioreactor. In another paper [24
], they modeled the biodegradation of 1,4-dioxane in the presence of THF using a cometabolism model to describe the stimulating effects of THF on 1,4-dioxane. In our study, any stimulating effects of THF or other structural analogs on the biodegradation of 1,4-dioxane were not observed in batch experiments. It could be that the initial bacterial population in our experiments was relatively high and that the additional growth effect by the inhibiting compounds may not have been significant. For a continuous flow process, it could very well be that the presence of fast-degraded structural analogs may effectively increase the population of 1,4-dioxane-degrading bacteria, stimulating the biodegradation of 1,4-dioxane. The stimulating effects in terms of increasing the 1,4-dioxane-degrading population are likely to partially offset their inhibitory effects observed in this study. Determination of the overall effects for a continuous-flow system requires more detailed quantitative modeling analysis. Additional biodegradation studies and modeling analysis for a continuous flow system are under progress using the raw wastewater.