Effects of Additional Carbon Sources in the Biodegradation of 1,4-Dioxane by a Mixed Culture

1. Introduction

1,4-Dioxane is a cyclic ether commonly found in textile wastewaters. It is typically formed as a byproduct during the production of organic fibers from terephthalic acid and ethylene glycol [1,2,3]. Although 1,4-dioxane is not acutely toxic, it is classified as a probable human carcinogen. In Korea, 1,4-dioxane has been detected at concentration levels up to few hundreds ppm in one of the major rivers, where many textile industries are located nearby. This brought up concerns about 1,4-dioxane, prompting an introduction of new regulations on the compound. There are also reports showing the common presence of 1,4-dioxane in wastewater treatment plant effluents [4,5,6,7]. However, it is not easy to treat 1,4-dioxane in wastewater because of its cyclic structure and ether linkage [8,9]. The compound is known to biodegrade very slowly [10,11,12,13,14], and the hydraulic retention time of typical wastewater treatment facilities may not be sufficient for the complete biodegradation of 1,4-dioxane. However, the biological treatment of 1,4-dioxane appears to be a good treatment option, due to the unavailability of alternative treatment methods. Due to its high solubility and low vapor pressure (40 mmHg at 25 °C), physico-chemical treatment processes such as air stripping and adsorption may not be efficient. The presence of activated sludge treatment facilities in most 1,4-dioxane discharging sources is another advantage of biological treatment. Understanding the biodegradation behavior of 1,4-dioxane is crucial to optimizing biological treatment processes for its effective and efficient removal from wastewater.

The biodegradation behaviors of 1,4-dioxane have been explored for both pure culture [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23] and mixed culture [4,24,25,26]. These studies showed that there are some bacteria that utilize 1,4-dioxane as a sole energy and carbon source. Applications of biological treatment processes for 1,4-dioxane removal have also been reported [5,27,28,29,30,31,32]. The slow degradation rate appears to be the main limitation of biological treatment regardless of the bacteria used. Cometabolic degradation with structural analogs such as tetrahydrofuran (THF) was also reported [24,33]. However, most studies are conducted for relatively clean waters such as groundwater and surface water, limiting their applicability to industrial wastewater [34,35,36,37,38,39,40]. The biodegradation potential of 1,4-dioxane was evaluated in different natural bacteria sources, indicating that 1,4-dioxane-degrading bacteria are not ubiquitously distributed in natural environment; an interesting conclusion of this study was that 1,4-dioxane-degrading bacteria do not always exist in contaminated sources [36]. In particular, the presence of other carbon sources in industrial wastewater may have significant effects on the biodegradation of 1,4-dioxane. Typically, 1,4-dioxane in wastewater coincides with relatively high level of biological oxygen demand (BOD) materials. The degradation of 1,4-dioxane may be retarded due to the presence of easily degradable organic materials. In addition, there may be some structural analogs coexisting in the wastewater, hindering the degradation of 1,4-dioxane. So far, studies on the effects of other carbon sources on the biodegradation of 1,4-dioxane are sparse.

In this study, the biodegradation of 1,4-dioxane by a mixed consortium of bacteria enriched from textile wastewater was studied in batch experiments. The effects of various carbon sources on the biodegradation rate of 1,4-dioxane were investigated. The biodegradation rate of 1,4-dioxane in textile wastewater was determined and explained by the presence of structural analogs such as 2-methyl-1,3-dioxolane and 1,4-dioxene.

2. Materials and Methods

2.1. Enrichment of Mixed Consortium of 1,4-Dioxane-Degrading Bacteria

A sludge was taken from a textile wastewater treatment plant that has been known to discharge 1,4-dioxane for years. The sludge was cultured at room temperature in basic salt media (BSM) containing 3240 mg/L K₂HPO₄, 1000 mg/L NaH₂PO₄·H₂0, 2000 mg/L NH₄Cl, 200 mg/L MgSO₄·H₂O, 12.6 mg/L FeSO₄·H₂O, 3 mg/L MnSO₄, 3 mg/L ZnSO₄·7H₂O, and 1 mg/L CoCl₂·6H₂O. Two hundred milligrams per liter of 1,4-dioxane was added as a sole carbon source, and the biodegradation of 1,4-dioxane was monitored by measuring the soluble chemical oxygen demand (COD) of the culture. Once the soluble COD was depleted, a small portion of the culture was seeded into a fresh BSM solution with 50 mg/L of 1,4-dioxane and the process was repeated several times.

2.2. Determination of Kinetic Coefficients for 1,4-Dioxane Biodegradation

Fifty-milliliter solutions of varying concentrations of 1,4-dioxane were prepared in a series of 100 mL flasks and inoculated with 10 mL of the seed culture, which was enriched in BSM, and the flasks were incubated in a shaking incubator at 25 °C. At times, small samples were taken from each flask and analyzed for 1,4-dioxane after filtration by a 0.22 μm syringe filter. To determine the yield coefficient, the cell growth was also assessed by measuring mixed liquor volatile suspended solids (MLVSS) according to a standard method [41]. For all biodegradation experiments, the seed culture was initially incubated in a rotary shaker in 1000 mg/L of 1,4-dioxane until it reached the exponential growth phase to avoid any lag phase or adaptation period in the early phase of biodegradation. Then, the culture was washed twice by centrifugation with sterile BSM and re-suspended before it was used for inoculation.

Based on the Monod equation, the degradation rate of 1,4-dioxane in a batch reactor can be expressed as Equation (1) [42], assuming the effect of cell decay is negligible:

$$\frac{dS}{dT}=q_{max}\frac{S}{K_s+S}\left[X_a^0+Y\left(S^0-S\right)\right]$$

(1)

where K_s is the half saturation constant, q_max is the maximum specific substrate utilization rate, Y is the cell yield, X_a⁰ is the initial biomass concentration, and S⁰ and S represent 1,4-dioxane concentration initially and at time t, respectively. The integrated form of the equation is given as:

$$t=\frac{1}{q_{max}}\left(\frac{K_s}{X_a^0+Y{S}^0}+\frac{1}{Y}\right)ln\left(X_a^0+Y{S}^0-YS\right)-\left(\frac{K_s}{X_a^0+Y{S}^0}\right)ln\frac{S{X}_a^0}{S^0}-\frac{1}{Y}ln{X}_a^0$$

(2)

When kinetic coefficients were estimated, the biodegradation of 1,4-dioxane with different biomass concentrations was determined to compare with the Monod equation.

2.3. Effects of the Presence of Other Carbon Sources on 1,4-Dioxane Biodegradation

Prior to the biodegradation experiments, the wastewaters from which the 1,4-dioxane-degrading mixed consortium was obtained were analyzed using GC/MS to identify any potentially inhibiting compounds other than 1,4-dioxane. Two cyclic ethers, 2-methyl-1,3-dioxolane and 1,4-dioxene, were confirmed to be present in the wastewater. Figure 1 shows the schematic diagram and hydraulic retention time (HRT) of textile wastewater treatment. The characteristics of the textile wastewater were summarized in

Table 1. The characteristics of the textile wastewater.

Characteristic	Value
2-Methyl-1,3-dioxolane (mg/L)	335.49–1082.8
1,4-Dioxene (mg/L)	76.27–132.12
1,4-Dioxane (mg/L)	188.7–320.6
CODcr (mg/L)	1250–2882
Suspended solids (mg/L)	106–178
pH	8.4–8.7

.

The biodegradation of 1,4-dioxane in batch flasks was determined in the presence of other carbon sources such as dextrose, THF, 2-methyl-1,3-dioxolane, and 1,4-dioxene. A series of 100 mL flasks containing 50 mL BSM, 1,4-dioxane, and other carbon sources was inoculated with the seed culture. Three duplicated flasks containing 1,4-dioxane with 20, 75, and 150 mg/L of THF were used to evaluate the influence of THF. In addition, 20, 100, and 500 mg/L of glucose were added to the three flasks to investigate the effect of easily degradable organic compounds on 1,4-dioxane degradation. The flasks were incubated in rotary shaker until the late exponential growth phase, at which time the cells were harvested, washed by centrifugation (5000 rpm, 5 min, 20 °C) with sterile BSM, and re-suspended in sterile BSM to obtain optical densities at 600 nm (OD₆₀₀) of 0.01–1.5. The flasks with 75 and 150 mg/L of THF as a sole carbon source were also investigated for comparison. The solutions of 200, 600, and 1000 mg/L were used for 2-methyl-1,3-dioxolane and 200 mg/L was used for 1,4-dioxene. The test was done in duplicate with biotic and abiotic controls.

2.4. Analytical Method

The concentration of 1,4-dioxane during incubation was measured by a Agilent 6890 gas chromatograph (Hewlett–Packard Company, Wilmington, NC, USA) equipped with a 5973 mass spectrometer with a HP-5MS (30 m × 0.25 mm I.D. × 0.25 μm) fused-silica capillary column using a following liquid–liquid extraction (LLE). Ten milliliters of the filtered liquid sample was added to the 100 mL separation funnel and extracted twice by 5 mL of methyl chloride for 5 min. After passing through the sulfuric anhydride trap to remove water, 2 mL of the methylene chloride extract was used for the analysis. The column temperature program was set at 100 °C hold for 1 min, 15 °C/min to 160 °C, and 5 °C/min to 300 °C hold for 7 min. The GC injector was held isothermally at 280 °C with a splitless period of 3 min. Helium (99.999%, flow of 1 mL/min) was used as a carrier gas at a flow rate of 1 mL/min using electronic pressure control. Soluble COD was also measured to double-check the 1,4-dioxane data using a DR/2010 Portable Data Logging Spectrophotometer (HACH, Loveland, CO, USA). The concentration of bacteria was measured as MLVSS.

3. Results and Discussion

3.1. Determination of Kinetic Parameters

The half saturation constant (K_s) and the maximum specific substrate utilization rate (q_max) 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 K_s, q_max, 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 [33,43,44]. 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 q_max 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 13b).

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.

4. Conclusions

To study the biological treatability of 1,4-dioxane-containing wastewater, the effects of coexisting carbon sources such as easily degradable organics and structural analogs on 1,4-dioxane biodegradation were determined using a mixed culture enriched for 1,4-dioxane. The biodegradation of 1,4-dioxane was not affected by the presence of dextrose, indicating no substrate competitiveness on the specific enzymes involved in 1,4-dioxane biodegradation. On the other hand, competitive inhibition was observed with THF, 2-methyl-1,3-dioxolane, and 1,4-dioxene, showing almost complete inhibition when the concentrations of the compounds were relatively high. All three compounds appeared to have much higher affinity over 1,4-dioxane for the enzymes acting on the rate-limiting step of 1,4-dioxane biodegradation. The inhibiting effects were also confirmed from the biodegradation experiments with the raw wastewater from a textile manufacturer containing a relatively high concentration of 2-methyl-1,3-dioxolane. It is likely that a much longer retention time is required for the proper treatment of 1,4-dioxane when these structural analogs are present in the wastewater. When treating 1,4-dioxane-containing wastewater, the presence and levels of other cyclic ethers and their inhibitory effects should be carefully estimated and reflected in the design of treatment systems.