Comparison of time-dependent induction of our three recombinant luminescent E. coli strains with toluene
Figure 1 illustrates the construction of the three recombinant plasmids. According to the preliminary experiment, the logarithmic growth phases of the three recombinant E. coli strains occurred from 6 to 15 h of incubation, and the relationship between OD of bacterial growth and RLU (Relative Light Unit) emitted from the three recombinant E. coli strains was linear from 8 to 14 h of incubation. Accordingly, the inoculation time of the three recombinant E. coli strains for the subsequent experiment was set as 12 h after incubation. Figure 2 presents a comparison of the time-dependent induction of luminescence emitted from T3-lux-E. coli, SP6-lux-E. coli, and T7-lux-E. coli caused by different toluene concentrations. As shown in Figure 2, the induction of luminescence caused by different toluene concentrations occurred time-dependently, regardless of the promoter type. The luminescence intensity continuously increased, leveled off, and then began to decrease considerably during incubation, all potentially due to the biochemical nature of the reporter gene lux .
The results demonstrated that luminescence was stable and the greatest at 2–2.5 h after incubation for T3-lux-E. coli and T7-lux-E. coli or 1–1.5 h after incubation for SP6-lux-E. coli. The time was equal to or shorter than that previously reported for the lux-based bioluminescent bioreporter P. putida TVA8 (2 h) and luminescence bacterial biosensors without the T7 promoter (3 h) for toluene measurement [6, 21]. Therefore, on average, 20-min consecutive measurements were recorded when T3-lux-E. coli and T7-lux-E. coli were cultured for 2 h and when SP6-lux-E. coli was cultured for 1 h. The maximum average luminescence induced by 200 μM toluene for T3-lux-E. coli, SP6-lux-E. coli, and T7-lux-E. coli was 1020 ± 20, 510 ± 10, and 2120 ± 60 RLU, respectively. Moreover, at the same toluene concentration, the signal intensity of luminescence decreased as follows: T7-lux-E. coli > T3-lux-E. coli > SP6-lux-E. coli. However, SP6-lux-E. coli had the shortest stable period for luminescence induction. Previous research demonstrated an increase in bioluminescence emission by fusing the T7 promoter to control expression of the lux operon .
Effects of culture conditions on luminescence
The effects of incubation temperature and ionic strength on the induction of luminescence biosensors for toluene were evaluated according to practical considerations. Figure 3A illustrates the effects of incubation temperature on the luminescence induced by 100 μM toluene for T7-lux-E. coli. The experimental results demonstrated the optimal temperature range of luminescence for T7-lux-E. coli to be 30–37 °C, with nonsignificant differences (p > 0.05). Similar results were observed for T3-lux-E. coli and SP6-lux-E. coli. Moreover, luminescences of the three recombinant E. coli strains at 20 and 40 °C were 12.1%–15.3% and 24.4%–26.8% lower than those at 37 °C, respectively. The effect of high temperature on the luminescence of the recombinant E. coli strain was more noticeable, a result attributable to the physiological characteristics of the E. coli . Thus, subsequent experiments were performed at 37 °C for all three recombinant E. coli strains.
Figure 3B shows the effects of ionic strength on the luminescence of the recombinant E. coli with the T3, SP6, or T7 promoter that were induced by 100 μM toluene. The results demonstrated almost no effect of different ionic strengths on the luminescence for SP6-lux-E. coli, but the ionic strength had greater effects on that of T7-lux-E. coli. When the ionic strength was 0.55 M, the luminescence of T7-lux-E. coli decreased by 12.5% ± 0.6%. This inconsistency among the recombinant E. coli with different promoters was presumed to be related to promoter structure and composition, which determine the strength of various types of promoter–target DNA bonds . Additional experiments to investigate these differences are planned. In general, the ranges of ionic strengths of groundwater, river water, seawater, and polluted water are 0.01–0.02 M, 10−3–10−2 M, 0.45–0.55 M, and >10−2 M, respectively. Thus, SP6-lux-E. coli is suitable for application in various water environments (groundwater, river water, and seawater), whereas T7-lux-E. coli is suitable for use in low ionic strength environments.
Effects of coexisting carbon sources, intermediates, and toluene analogs on luminescence
The xyl genes of the Pseudomonas putida TOL plasmid encode the genetic information required for the degradation of toluene and related aromatic compounds. The xylR and xylS genes of the xyl structural genes encode the regulatory proteins of the catabolic operons, whereas the XylR protein is the master regulator of TOL plasmid catabolic operons for the metabolism of toluene . Transcription of the operon is positively regulated by the XylR/XylS protein activated by toluene, xylenes, or their alcohol catabolic products . Figure 4A illustrates the effects of coexisting carbon sources at 100 μM on the luminescence of T7-lux-E. coli and SP6-lux-E. coli. The tested chemicals are considered potential inhibitors or activators (indirect or direct inducers) of xylS and xylR and may deviate significantly or have an additive effect in relation to theoretically expected effects, calculated on the basis of individual chemicals [12, 33, 34]. The current results demonstrated that the coexistence of lactate or glycerin with toluene induced greater luminescence than did toluene alone. Lactate at 100 μM increased luminescence by 21% ± 8.6% for T7-lux-E. coli and 20.3% ± 5.1% for SP6-lux-E. coli, while glycerin increased by 14% ± 1.8% for T7-lux-E. coli and 13.5% ± 3.5% for SP6-lux-E. coli, respectively. The increased luminescence disappeared when the concentrations were below 70 μM (lactate) or 85 μM (glycerin). By contrast, the coexistence of acetate with toluene induced lower luminescence than did toluene alone; luminescence decreased by 32% ± 1.5% for T7-lux-E. coli and 32.5% ± 2.9% for SP6-lux-E. coli. However, for other chemicals, the coexistence had negligible effect on the detection of toluene by T7-lux-E. coli and SP6-lux-E. coli.
Figure 4B illustrates the effects of the benzoate concentration on the luminescence of T7-lux-E. coli and SP6-lux-E. coli. Benzoate is the most important metabolite produced during toluene biodegradation , which may affect XylS expression ; thus, we evaluated the effect of the benzoate concentration on the luminescence of T7-lux-E. coli and SP6-lux-E. coli. The results demonstrated that a high benzoate concentration could induce higher luminescence than did toluene alone, as detected using T7-lux-E. coli and SP6-lux-E. coli. Although 50–150 μM benzoate did not affect luminescence, 250–300 μM benzoate increased luminescence by 26% ± 3.5% for T7-lux-E. coli (25.4% ± 1.8% for SP6-lux-E. coli) and 34% ± 2.8% for T7-lux-E. coli (33.8% ± 1.8% for SP6-lux-E. coli), respectively. In other words, the effect of low concentrations of benzoate on luminescence was limited when toluene was detected by T7-lux-E. coli or SP6-lux-E. coli.
Figure 4C illustrates the effects of toluene analogs and their concentrations on the luminescence of T7-lux-E. coli and SP6-lux-E. coli. The results demonstrated that the various concentrations of o-xylene and p-xylene had negligible effects on toluene detection by the recombinant E. coli biosensor; moreover, even when 250 μM o-xylene was used, only 4.15%–4.30% increase in luminescence was observed. However, 250 μM m-xylene and 250 μM benzene induced T7-lux-E. coli or SP6-lux-E. coli to produce relatively high luminescence (12.3%–12.5% and 14.3%–14.6%, respectively). By contrast, the effect of the toluene analog concentration of ≤200 μM on toluene detection was limited (<8%). The effect of the synergistic mode was far lower than that observed in the P. putida mt-2 KG1206 biosensor .
Taken together, these results illustrate that our recombinant luminescent biosensor possesses high selectivity and specificity when detecting a group of analytes with similar chemical structures. Because the included chemicals mainly affect the regulatory genes xylS or xylR, but not the T3, SP6, or T7 promoter, their effects on the magnitude of luminescence among all three recombinant E. coli biosensors were similar . Figure 4 exemplifies the cases of T7-lux-E. coli and SP6-lux-E. coli.
Relationship of toluene concentration with luminescence
The function of these promoters (T7, T3, SP6) is to make the downstream reporter gene (lux) more strongly expressed. Therefore, xylR is first induced in the presence of toluene and activates gene expression, then the promoters and reporter gene (lux) follow. Under optimal operating conditions, we determined the relationships between the toluene concentration and the luminescence of the three recombinant E. coli strains. Two sets of linear relationships were observed between the toluene concentration and luminescence at different concentration ranges. Figure 5A presents a set of regression equations for the toluene concentration and the luminescence of T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli when the toluene concentration was 10–500 μM: y = 6.140x + 724.9, y = 3.233x + 302.2, and y = 1.560x + 154.9, respectively. Figure 5B presents another set regression equations for T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli when the toluene concentration was ≤10 μM: y = 40.515x + 46.9, y = 11.666x + 24.5, and y = 7.868x + 17.6, respectively. The coefficients of determination for these equations was high (>0.99), indicating their reliability. The concentration-dependent differences in these linear relationships may have been due to differences in promoter characteristics . Moreover, for T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli, the LODs for toluene were 0.05, 0.2, and 0.5 μM, respectively. Therefore, T7-lux-E. coli was the most sensitive. Willardson et al. (1998) constructed a bacterial biosensor with the reporter gene luc, Casavant et al. (2003) constructed a site-specific recombination-based biosensor with tbuA1UBVA2C promoter, Li et al. (2008) constructed a lux-based bacterial biosensor, Zeinoddini et al. (2010) constructed a aequorin-based E. coli biosensor, Zhong et al. (2011) constructed a monooxygenase biosensor, and Ray et al. (2018) constructed a protein-based biosensor; their LODs for toluene were 10, 0.2, 7.5, 1, 3, and 3.3 μM, respectively [13, 20–22, 37, 38]. Compared with the aforementioned biosystems, T7-lux-E. coli has lower LOD (0.05 μM), indicating acceptable sensitivity. To develop a biosensor for detecting toluene, reporter genes such as luc, lux, and aequorin were often constructed downstream of the degradation gene. However, these biosensors could not measure trace levels of toluene contamination in wastewater. To improve the LOD, a promoter (T7, T3, or SP6) was inserted between the degradation gene and reporter gene. To our knowledge, little has been reported on applying this strategy to regulate the expression of the reporter gene and improve the LOD of a biosensor for toluene. In conclusion, the novel plasmid or biosensor with low LOD constructed here exhibited high potential for measuring bioavailable toluene.
Hence, on the basis of the aforementioned reliable equations or the calibration curve for 0.05–10 or 10–500 μM toluene, the toluene concentration in the water samples can be rapidly determined. In addition, the broad detection ranges of T7-lux-E. coli indicate that it is a practical toluene measurement tool.
To evaluate the reproducibility of the biosensors for detecting toluene, T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli were tested under identical conditions by using TMM containing 10 μM toluene. Relative standard deviation (RSD) for T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli was 4.3%, 5.1%, and 5.8%, respectively (n = 10). Batch-to-batch variation was also tested by comparing the luminescence from the five sets, which was tested using TMM containing 10 μM toluene, and the RSD for T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli was 6.2%, 6.5%, and 9.4%, respectively. These results are comparable to the reproducibility reported for two induction-based toluene biosensors: RSD = 9.5% for n = 3 (with 21.7 μM toluene) and RSD = 7.4% for n = 8 (with 92 μM toluene) [38, 39]. Thus, our recombinant luminescent E. coli biosensors demonstrated operational stability. Similar results were obtained when these biosensors were applied for measuring 10 μM toluene after a 3-month cryogenic storage period.
Toluene measurement in groundwater and river water by using our three recombinant luminescent E. coli biosensors
Most luminescent biosensors have been applied for measuring toluene availability in artificial wastewater, but few have been applied in actual wastewater. Table 1 summarizes the measured toluene concentrations in seven groundwater samples and three river water samples using our three recombinant luminescent E. coli biosensors and the standard GC–MS method. The results demonstrated that the toluene concentration determined using our biosensors and through GC–MS demonstrated excellent correlation (r2 > 0.998); moreover, the deviation between the toluene concentrations measured through GC–MS and those measured using T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli was −14.3% to 9.1%, −10.7% to 26.7%, and −3.6% to 4.2%, respectively. Considering the measurement ranges and accuracy, T7-lux-E. coli provided the accurate and reliable toluene measurement in these aqueous matrices. However, under appropriate toluene concentration ranges, SP6-lux-E. coli could be the best biosensor in terms of accuracy, and its genetic assembly is relatively less susceptible to environmental interference . The measurement deviation of T7-lux-E. coli and SP6-lux-E. coli were comparable to that (−16.7% to 7.5%) of electrochemical inhibition bacterial sensor array for toluene detection . Taken together, these results indicate that the developed recombinant luminescent bacterial biosensors can determine toluene concentration in different water bodies.