Influence of Nonionic Surfactants on Fungal and Bacterial Degradation of Hexane

2.1 Chemicals

Hexane (99% purity, Fisher Scientific) was used as received. Four nonionic surfactants, Brij 30 (B30), Brij 35 (B35), Triton X-100 (TX100), and Tween 80 (T80), were purchased from Sigma–Aldrich and used without further purification. The properties of the tested surfactants are outlined in Tab. 1, as reported in previous studies. The hydrophile–lipophile balance (HLB) defines the proportion of hydrophilic/lipophilic groups in a surfactant molecule 22. A low HLB value indicates a hydrophobic characteristic for surfactants, and vice versa. The critical micelle concentration (CMC) used in this study was 12.7, 110.2, 143.8, 17.0 mg L⁻¹ for B30, B35, TX100, and T80, respectively 11, 19, 20, 23-30.

Table 1. Characteristics of selected surfactants

Trade name	Structurea)	Formulaa)	MWa)	HLBb)	CMC (mg L−1)c)	Molar ratiod)
B30		C12H25 (OC2H4)4OH	362	9.5	12.7	5.03 × 10−4
TX100		C8H17C6H4(OC2H4)10OH	625	13.5	143.8	4.04 × 10−5
B35		C12H25 (OC2H4)23OH	1200	16.9	110.2	2.26 × 10−6
T80		C18H37C6H9O5(OC2H4)20OH	1309	15.0	17.0	3.93 × 10−4

• a) Molecular information of nonionic surfactants tested refers to Laha and Luthy 20, Grimberg et al. 23, Prak and Pritchard 27, Tiehm 28, and Guha and Jaffe 40.
• b) HLB values refer to Griffin 22, Grimberg et al. 23, and Slinde and Flatmark 46.
• c) CMC values are selected based on previously reported values 11, 19, 20, 23-30.
• d) Molar ratio = (molar of hexane solubilized at CMC −  molar of hexane solubilized without surfactant)/molar of surfactant at CMC.

2.2 Solubility tests

Aqueous hexane solubility tests were conducted in 125 mL serum bottles (Wheaton Industries, NJ) with aluminum cap and septum. Stock surfactant solutions (0, 0.5, 1.0, 2.0, 5.0, and 10.0 CMC) were prepared and 50 mL was aliquoted into three replicate serum bottles (N_total = 72). To introduce hexane for solubility assessment, gas-phase hexane was generated in a static dilution bottle (Kimble & Chase, NJ) at room temperature and then transferred by a gas tight syringe (Valco Instruments, TX) into serum bottles, providing an initial 250 ppmv headspace concentration 9. Then, the serum bottles were placed into a tumbler at 12 rpm for mixing. Sampling time was at 1, 3, 6, 12, 24, 36, 48, 72, and 96 h.

2.3 Fungal and bacterial culture preparation

Both fungal and bacterial cultures were obtained from a TBAB system at the University of Cincinnati. Effluent samples containing fungal and bacterial biomass were collected during backwashing period of the filters. Zehraoui et al. 31 characterized the species of microorganisms in the TBAB system. They identified Fusarium spp. and A. niger as the dominant species for fungi and Mycobacterium sp. for bacteria using the 16S rDNA and 18S rDNA PCR and sequencing analyses 31. Collected biomass samples from each filter were centrifuged and grown in batch reactors at pH 4 (fungi promoting) and pH 7 (bacteria promoting), respectively, until steady state growth was achieved. Gas phase hexane was added to the reactors as the sole carbon source using a syringe pump (KD Scientific, MA) at a loading rate of 5.37 g m⁻³ · h⁻¹ through a gas diffuser submerged in the reactor. A micronutrient solution of mineral salts and buffered nutrients was also added to the reactors following the chemical composition by Hassan and Sorial 13. The detailed chemical composition of the micronutrient solution is provided in Supporting Information Tab. S1. After reaching steady-state growth, the biomass from both fungal and bacterial reactors was harvested and washed with phosphate buffer solution (pH 4 and 7 for fungi and bacteria, respectively), so frozen culture stock solutions could be prepared for microcosm tests.

2.4 Microcosm study

In order to elucidate the role of nonionic surfactants on hexane degradation kinetics, microcosm tests were conducted using 125 mL serum bottles. A working volume of 50 mL was utilized for the tests, which included microbial seed culture, mineral salts, nutrients, and surfactant at various concentrations (0, 0.5, and 5.0 CMC). Student's t-test conducted using JMP statistical software suggested that the difference in gas phase hexane solubility at 0.5 and 5.0 CMC was significant. Solution pH was adjusted to pH 4 for fungal biomass experiments and pH 7 for bacterial biomass degradation tests and 1 mL of culture stock solution was added to each bottle to initiate the trials. Then, the prepared sample bottles were placed in a rotary tumbler at 12 rpm and the gas-phase hexane concentrations in the bottles were monitored every 2 days for 14–18 days according to previously reported acclimation times for hexane degradation in TBAB systems 9, 14, 31, 32. All microcosm tests were conducted at room temperature (20°C) to be consistent with the common operational temperature of TBAB in other studies 9, 13, 14.

2.5 Analysis

A gas chromatograph (Hewlett-Packard 6890) equipped with a mass selective detector and HP-624 column (30 m × 250 μm × 0.25 μm) was used to measure the concentration of hexane in the gas phase. For the analysis, the flow rate of the carrier gas (helium, 99.999%) was 6.7 mL min⁻¹. The set point temperature of the heater was at 260°C and pressure was at 2.82 psi. Detector and injector temperatures were held at 250 and 120°C, respectively. Standard gas-phase solutions for hexane were prepared by spiking different amount of liquid hexane into the static dilution bottles. Once the hexane completely vaporized in the dilution bottles, the gas-phase sample was transferred to the GC/MS injector using a 0.5 mL gas tight syringe. Evaluation of serum bottle hexane concentrations followed the same injection method.

3.1 Hexane solubility kinetics

Hexane equilibrium between aqueous and gas phase was reached within 10 h in the presence of all tested surfactants (Supporting Information Figs. S1–S4). Figure 1 shows the equilibrium concentration of hexane in the head space (ppmv) demonstrating an increased solubility of hexane with increasing surfactant concentration. A lower hexane concentration in the headspace represents a greater hexane concentration in the aqueous phase (both aqueous bulk and micelle). The headspace hexane concentration stabilized at 188.5 ppmv after reaching equilibrium between gas phase and water. The presence of B30, TX100, and T80 significantly decreased the headspace hexane concentration from 188.5 ppmv to 166.8, 147.3, and 149.5 ppmv, respectively, whereas B35 did not affect the dissolution of gas-phase hexane. It was noted that the apparent solubility of hexane did not increase proportionally as surfactant concentrations exceeded CMC, where these results do not follow the trend observed for other hydrophobic organic compounds (e.g., polycyclic aromatic hydrocarbons) in surfactant solutions above CMC 27, 33. Before partitioning into the micellar cores of surfactants and achieving pseudo-solubilization, mass transfer of the hexane through the air–water interface must occur between the headspace and aqueous solution in the serum bottle. Thus, the key mass transfer limitation in enhancing aqueous hexane solubility in the presence of surfactants appears to be related to slow transport of hexane across the air–water interface as limited by its high vapor pressure and corresponding Henry's law constant, which results in the nonlinear correlation between apparent hexane solubility and surfactant concentration 34. In order to evaluate the surfactant effect on hexane solubility, the following first order dissolution model adapted from Grimberg et al. 23 was used to fit the dissolution data of gas-phase hexane:

(1)

where C is the hexane concentration in the aqueous phase (M⁻¹), C_eq is the equilibrium concentration in the aqueous solution (M⁻¹), k_obs is the observed dissolution rate (h⁻¹), and t is the time (h). The nonlinear regression analysis, performed by OriginPro 7.5 software, showed that the proposed model is well fitted to the observed dissolution data with R² > 0.91 (p < 0.05). Figure 2 shows the changes in dissolution rates, k_obs, of gas-phase hexane with increased surfactant concentrations. At <2 CMC, a significant acceleration of hexane dissolution was observed with surfactant addition. However, at >2 CMC, the change in the dissolution rate of hexane was greatly diminished except for B30.

Table 1 includes the molar solubility ratio of tested surfactants (B35 < TX100 < T80 < B30) at their respective CMC. The ratio was determined by calculating the molar concentration differences of hexane solubilized in a surfactant solution at CMC and in a solution without a surfactant divided by the molar concentration of the surfactant solution. Differences in solubility enhancement by the tested surfactants were attributed to their unique molecular structures which affects hexane partitioning into the micelles. As shown in Tab. 1, B30, TX100, and B35 all have one hydrophilic polyoxyethylene sequence and a short tail of hydrophobic alkyl chain. T80, on the other hand, possesses several polyoxyethylene chains and a long hydrophobic alkyl tail. The solubility enhancements by the former three surfactants correlated inversely with the HLB values. A higher HLB value indicates a more hydrophilic micelle and thus, a smaller hydrophobic core in the micelle, resulting in reduced hexane partitioning into the micelle. Grimberg et al. 23 observed the same solubility trend when evaluating surfactant HLB values for hydrophobic phenanthrene. Despite this trend, T80 has a relatively high HLB value of 15 (Tab. 1); however, its long hydrophobic alkyl chain can result in more hexane partitioned into the micellar phase, therefore, facilitating the solubilization of hexane.

The effect of surfactants on the dissolution rate of gas-phase hexane is controlled by two steps: mass transfer and saturation 23. The number of saturation sites is a linear function of the surfactant concentration. Therefore, as described previously, the observed nonlinear relationship between solubility rate of hexane and surfactant concentration was due to the insufficient mass transfer of hexane from headspace to solution. The higher dissolution rates of hexane in B30 and TX100 than those of B35 and T80 are probably attributed to the relatively smaller molecular size of the surfactants, which is related to a higher diffusivity in water resulting in faster mass transfer process 35.

3.2 Microcosm study

The effects of nonionic surfactants on fungal and bacterial growth utilizing hexane were investigated with a serum bottle-based microcosm study. The microcosm study results showed that the amount of hexane in the headspace decreased rapidly in 9 days and then remained unchanged for both cultures (Supporting Information Fig. S5; selected surfactants with high hexane removal are shown for representative data). Figure 3 shows the biomass growth in the serum bottles and corresponding hexane removal per unit biomass in the presence of tested surfactants. After injection, the gas phase hexane firstly dissolved into the aqueous solution resulting in the reduction of headspace hexane concentration. Therefore, hexane removal due to biodegradation was calculated by subtracting the reduced concentration at designated operation time from the solubility concentration at equilibrium. Overall, the fungi and bacteria responded differently to the addition of tested surfactants, despite optimized growth pH. For example, the hexane removal per unit mass of fungal culture without surfactant was 3.2 mg g⁻¹, with a maximum removal of 4.5 mg g⁻¹ in the presence of 0.5 CMC B30 during the experimental period. For bacterial degradation, the final hexane removal at pH 7 in the absence of surfactant was 22.3 mg g⁻¹, whereas addition of 0.5 CMC B30, B35, or TX100 increased the removal up to 58.4 mg g⁻¹. Overall improvement in degradation was attributed to the surfactant-assisted mass transfer of hexane into the aqueous phase for bio-uptake. The difference in hexane removal between fungi and bacteria could be due to the lower growth rate of fungi as compared to bacteria, which is consistent to previous observations 36. Another possible reason for the low hexane removal was temperature effects. Iranmanesh et al. 3 recently reported an optimum temperature of 36.5°C for fungal degradation of hexane. The removal was reduced to 32.6% at a temperature of 25°C. The microcosm tests in this study were conducted at room temperature (20°C) which likely diminishes fungal metabolic activity.

For fungal degradation of hexane, the results in Fig. 3a suggest that 0.5 CMC B30 and T80 increased the hexane removal while B35 and TX100 showed no improvement to the degradation process. Higher fungal growth (7.0–40.0 mg L⁻¹ higher biomass than control) was observed for all surfactants except for TX100 as it exhibited toxic effects to the fungal culture 37. However, TX100 can be a good candidate when seeking for comparable hexane degradation at a much lower biomass accumulation. It is further noted that B35 with the lowest observed solubility enhancement yielded the highest biomass growth, followed by B30 and T80. This may indicate that fungi utilized the surfactants, especially B35, as carbon source 38 resulting in greater fungal growth 39. At 5 CMC, the hexane biodegradation and biomass growth (except for B35) were inhibited due to the toxicity of the surfactants as well as increased partitioning of hexane into surfactant micelle phase (Fig. 3b) 40. The large amount of surfactant molecules likely reshaped the cellular structures of fungi by interacting with its lipid component or reacting with enzymes and other essential proteins in the fungal cell 20, 41. As shown in Fig. 3b, the dysfunctional ability of TX100 and T80 for fungal growth was lower than that of B30 which, however, yields a comparable hexane removal to the control without surfactants. In contrast, fungi easily utilized B35 at high concentrations over hexane as carbon source in aqueous solution, leading to reduced hexane degradation. This preferential consumption of B35 as an abundant carbon source resulted in a slightly higher biomass growth than that of control in Fig. 3b.

Figure 3c shows that B30, B35, and TX100 at low concentrations (0.5 CMC) all enhanced the bacterial degradation of hexane. The increased hexane removal was attributed to increased mass transfer into the aqueous facilitated by the nonionic surfactants which worked as transfer terminals for hexane. Considering similar biomass growth in the presence of T80 compared to that of B30, the reduced hexane removal by T80 was possibly due to preferential utilization of T80 over hexane as a substrate for bacterial growth 42. The highest biomass yield under B35 was likely the result of consumption of the surfactant. In addition, TX100 showed facilitation, instead of inhibition, to both biomass growth and biodegradation. As compared to TX100, B30 promoted less biomass growth for the same removal efficiency, suggesting B30 is a better candidate for biofiltration systems for hexane removal with less backwashing. In Fig. 3d, the presence of surfactants at high concentrations (5 CMC) caused adverse effects on the bacterial biodegradation process. The adverse effects were more evident for B30 and TX100 even though both surfactants facilitated the gas-phase hexane dissolved in water. For B35, higher biomass growth was also observed, which again implies B35 was utilized by bacteria as a preferential substrate. A slightly increased biomass growth in the presence of T80 further confirms the utilization of T80 as substrate. The higher biomass yield with B35 and T80 subsequently consumed more hexane than that of B30 and TX100 by co-metabolism or biosorption 39. Since 5 CMC surfactants did not inhibit biomass growth, the effect of surfactant toxicity was excluded for bacterial degradation of hexane. The decreased hexane bioavailability under high surfactant concentration was possibly due to the increased partitioning of hexane into surfactant micelle phase as a function of micelle quantity, which limited direct mass transfer from the micelle to microorganisms. Guha and Jaffe 40 reported similar observations for the biodegradation of a model polycyclic aromatic hydrocarbon (i.e., phenanthrene). In their microcosm studies, they observed a slower degradation rate of phenanthrene with surfactant concentrations beyond CMC. They concluded that bioavailability of phenanthrene decreased with increasing surfactant concentrations due to mass transfer limitations of the partitioned phenanthrene from surfactant micelles to the bacteria.

3.3 Hexane degradation kinetics

The relationship between the biomass growth and substrate concentration (C) can be described by the Monod kinetics 43:

(2)

where μ is the biomass growth rate (day⁻¹), μ_max is the maximum growth rate (day⁻¹), and K_S is the half-saturation constant (the concentration supporting half maximum uptake rate) for growth (mg L⁻¹).

The concentration of substrate is inversely proportional to biomass growth 44, thus:

(3)

where v is the substrate utilization rate (mg L⁻¹ day⁻¹) and v_max is the maximum utilization rate (mg L⁻¹ day⁻¹).

When K_S >> C, Eq. 3 will be approximated to first-order kinetics 44:

(4)

Given the first-order rate constant:

(5)

then

(6)

Integrating Eq. 6, we can obtain

(7)

where C₀ is the initial concentration of hexane added into serum bottles.

Since the aqueous hexane concentration (<1.3 mg L⁻¹ at complete dissolution) was much smaller than the reported K_S value (16 mg L⁻¹) 45, the degradation result as a function of time was recorded and then fitted to the above linear relationship to obtain the pseudo first-order reaction rate constant k. Figure 4 shows the degradation kinetics of hexane by fungi (pH 4) and bacteria (pH 7) in the presence of tested nonionic surfactants. Overall, low surfactant concentrations increased the hexane degradation rates compared to controls without surfactants. However, the influence of high surfactant concentrations on degradation kinetics was more complicated. As compared to fungi, higher overall degradation rates were observed by bacteria which are possibly due to the larger cell population per biomass weight and the lower optimal metabolic temperature for bacterial growth.

In Fig. 4a, the comparable degradation rates between 0.5 CMC B35 and control were consistent with hexane removal in Fig. 3a. However, in Fig. 4b, 0.5 CMC B35 with the lowest solubility molar ratio (Tab. 1) exhibited the highest degradation rate for hexane by bacteria. The accelerated hexane degradation was likely due to the utilization of B35 as a carbon source which supported higher biomass growth and subsequently accelerated degradation of hexane. However, at higher concentrations, B35 outcompetes hexane for bio-uptake, thus diminishing the hexane biodegradation rate in comparison. The similar fungal degradation kinetics for 0.5 CMC B30, TX100, and T80 parallel the observed hexane solubility rates in Fig. 2. The same correlation was observed for bacterial degradation kinetics for B30 and TX100. As compared to surfactants below CMC, the reduced degradation rates at 5 CMC (except for T80 for bacteria) mirror the inhibited biomass growth in Fig. 3b and d indicating the adverse effect of high surfactant loading rates which may be associated with surfactant toxicity or reduced mass transfer of hexane due to strong partitioning of hexane into surfactant micelle phase. However, 5 CMC B30, TX100, and T80 still enhanced hexane degradation rates by fungi. As for T80, its higher level of hexane dissolution at 5 CMC (153.5 ± 1.3 ppmv in headspace) compared to that at 0.5 CMC (168.1 ± 1.4 ppmv in headspace) resulted in the faster degradation rate of hexane by bacteria. The slow hexane removal by 0.5 CMC T80 was consistent to the observation of reduced hexane degradation in Fig. 3c and d, although similar biomass growth was observed at 0.5 and 5.0 CMC T80 conditions, which again suggests potential utilization of T80 as a substrate for bacterial growth 42.

Overall, the surfactants (B30 and T80) tested in this study provided optimal hexane removal by fungal consortia at 0.5 CMC, whereas concentrations of B30 and TX100 below their CMC are better options for degradation of hexane if considering bacteria. Although B35 showed high hexane removal, B35 may not be the best surfactant for TBAB systems as it is easily consumed by fungi and bacteria producing high amount of biomass. Long-term monitoring of the fungal degradation of hexane in the presence of tested surfactants is needed. In addition, both fungal and bacterial consortia in the TBAB system will be present as biofilm grown on the supporting media of the system. Therefore, hexane degradation by the mixed consortium needs to be studied as attached biofilm and the influence of nonionic surfactant on biofilm formation and growth kinetics needs to be further determined with a fundamental understanding of interactions among microorganisms, VOCs, and surfactants.