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Effect of metal concentration on growth and luminescence of luminous bacteria strains isolated from golfo de Nicoya, Costa Rica

Luminescence in bacteria is catalyzed by luciferase. When these microorganisms are exposed to toxic substances, the bioluminescent enzyme system can be inhibited. The objective of this study was to analyze the potential that these microorganisms offer as native bioindicators of coastal marine pollution. The dynamics of luminescence intensity by visual classification and the effect of metal concentration on the growth and luminescence of 25 strains of luminescent bacteria, isolated during 2016 from seawater samples from the gulf of Nicoya, Costa Rica, was evaluated by the disk diffusion method. The sensitivity of each strain to different concentrations (0.1, 0.5 and 1 mg mL-1) of Cd, Cu, Cr, Pb and Zn was determined by its bioluminescent phenotype. In susceptible strains, a range of metal concentrations less than the growth inhibitory concentration affected the expression of luminescence. Strains with intense luminescence and defined zones of luminescence inhibition were considered to have greater potential as native bioindicators for monitoring environmental toxicity. More studies are required to determine the minimum concentrations that inhibit growth and luminescence with respect to the tested metals and other potentially toxic substances for the coastal marine ecosystems of Costa Rica.


INTRODUCTION
Most luminous bacteria, which are widely distributed in marine environments, are taxonomically classified as belonging to Aliivibrio, Vibrio, and Photobacterium genera from the Vibrionaceae family. Several of these bacteria emit high levels of light easily visible in laboratory cultures. Luminescence in these microorganisms is produced by the expression of lux genes, and the light emission reaction is catalyzed by luciferase (Dunlap & Urbanczyk, 2013).
Luciferase synthesis and luminescence in these bacteria is regulated by population density through a system called quorum sensing (Dunlap & Urbanczyk, 2013). Hence, there is a relationship between the emission of luminescence with cellular metabolism. The bioluminescent enzyme system can be inhibited by exposing bacteria to toxic substances, and the intensity of light decreases quickly. Measuring the light intensity of bacteria exposed to different concentrations of a substance is used to evaluate its toxicity (Ma et al. 2014). Sensitive, repeatable, and easy to transport luminous bacteria-based assays have been developed to detect environmental contaminants (Camanzi et al. 2011;Bolelli et al. 2016).
Therefore, luminous bacteria have been used as bioindicators in assessments of environmental pollution (Burga et al. 2012;Ma et al. 2014), toxicity evaluation of pharmaceutical wastewater (Yu et al. 2014) and toxicological tests to assess pollution caused by metals, pesticides, and antibiotics in aquatic systems (Ranjan et al. 2012;Shanware et al. 2013). Moreover, Bagordo et al. (2012) have used them to assess anthropogenic impacts on estuarine ecosystems. Luminous bacteria represent a powerful tool for the initial assessment of environmental samples or substances with unknown ecotoxicological or toxicological characteristics (Menz et al. 2013). In Costa Rica, there are no references of bioassays to assess the effect that toxic compounds might be causing in coastal marine ecosystems and, according to Diepens et al. (2014), promotion of ecotoxicological assessments in the country using native species is required. This study aimed to analyze the effect of metal concentration on growth and luminescence of luminous bacteria strains isolated from the golfo de Nicoya, Costa Rica, to generate base information about the application that these microorganisms have as native bioindicators of coastal marine pollution in the country.

MATERIALS AND METHODS
In 2016, twenty-five (25) luminous bacteria strains isolated from surface seawater samples collected at the golfo de Nicoya, Costa Rica, were used. These strains were isolated using Marine Agar (MA) (Difco®) and stored in Brain Heart Infusion (OXOID®) with 20% glycerol at -80°C until they were tested. These strains were numerically identified in a consecutive manner using the CL prefix.
Strains were characterized according to growth and luminescence intensity by visual classification following the methodology proposed by Kumar et al. (2015), with some variations. Every 2 h during a 14 h period, a record was made of growth and luminescence intensity of each strain inoculated by streaking swabs on MA plates (Dif-co®) from a cell suspension equivalent to a 0.5 McFarland standard in distilled water with 2% NaCl prepared from an overnight MA plate. At the end of the incubation period and according to the final luminescence intensity, the strains were classified as intense, reduced, or dim, based on the luminescence intensity scale defined in Fig. 1. The effect of metal concentration on growth and luminescence of these strains was assessed using the disk diffusion method. Each luminous strain was inoculated by streaking swabs in MA plates plates from a cell suspension equivalent to a 0.5 McFarland standard in distilled water with 2% NaCl. Thereafter that, 6 mm diameter Whatman #3 filter paper sterile disks were separately impregnated each with 10 µL of 0.1, 0.5, and 1 mg mL -1 metal ion solutions; namely, cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), and zinc (Zn), then they were aseptically dried at room temperature and placed on the agar surface. Disks impregnated with 10 µL of sterile distilled water were used as negative controls. Tests were performed duplicate. Plates were incubated in the dark at 30°C for 24 h.
After the incubation period, records were made concerning the diameter in mm of the growth inhibition zones (in the light) and the diameter in mm of the luminescence inhibition zones (in the dark). Every 2 h during 14 h of incubation, a photographic record was also made of the assay with Cd for CL4 and CL22 strains to illustrate the results. Photographs were taken in the dark with a 30 s exposure and under the same conditions (ISO500, F8) for comparison.
Mean difference and 95% confidence intervals were calculated between the diameters for inhibition zones of growth or luminescence by metals and metal ion concentration. Samples mean pair-wise comparisons were evaluated using a t-test (P < 0.05). In a case were the statistical assumption (t-test) was not meet the non-parametric Wilcoxon test was applied (P < 0.05). Results were analyzed and plotted using R (R Core Team, 2018).

RESULTS
The luminescence intensity scale is shown in Fig. 1. All strains grew 4 h after the incubation time started, and showed luminescence from 6 to 8 h after the start of the incubation (Fig. 2). The luminous strains assessed did not show growth and luminescence inhibition zones for the tested Cr concentrations. CL6, CL11, CL12, and CL19 strains showed dim luminescence (Fig.  2), and no inhibition zones (growth o luminescent) were observed (Fig. 5).
The diameters of the inhibition zones (growth and luminescence) were very similar, and the averages were used (Fig. 5). Most of the strains showed growth inhibition zones for 0.5 and 1 mg mL -1 concentrations of Cd, 1 mg mL -1 of Zn, and minimum diameters of 7 mm for 1 mg mL -1 concentration of Cu and Pb (Fig. 5).
Those as mentioned above showed luminescence inhibition zones concerning Pb at all assayed concentrations. These strains showed defined luminescence inhibition zones for 0.5 and 1 mg mL -1 Cd and Zn, respectively. Only CL15 and CL17 strains showed the same condition for 0.1 mg mL -1 Cd. CL9, CL10, and CL17 strains also  Fig. 3. Photographical record of the effect of 0.1 (A), 0.5 (B), and 1 (C) mg mL -1 cadmium on the variation of luminescence intensity for CL4 and CL22 luminous strains at different incubation times. D = control. Strain CL4 exhibits a degradation of luminescence around disks impregnated with Cd; strain CL22 shows defined growth inhibition zones (E) and luminescence inhibition zones (F) for 0.5 and 1 mg mL -1 Cd Fig. 3. Registro fotográfico del efecto de 0.1 (A), 0.5 (B) y 1 (C) mg mL -1 de cadmio en la variación de la intensidad de la luminiscencia para las cepas luminiscentes CL4 y CL22 a diferentes tiempos de incubación. D = control. La cepa CL4 muestra degradación de la luminiscencia alrededor de los discos impregnados con Cd, y la cepa CL22 muestra zonas de inhibición del crecimiento (E) y zonas de inhibición de la luminiscencia (F) definidas para concentraciones de Cd de 0.5 y 1 mg mL -1 Fig. 4. Luminescence reduction (•) by different concentrations (mg mL -1 ) of cadmium, copper, lead, and zinc ions on bacterial strains with reduced luminescence intensity isolated from the golfo de Nicoya, Costa Rica. Data for chromium ions not shown due to the absence of growth and luminescence inhibition zones Fig. 4. Reducción de luminiscencia (•) por concentración (mg mL -1 ) de cadmio, cobre, plomo y zinc de cepas bacterianas con intensidad de luminiscencia reducida aislada del golfo de Nicoya, Costa Rica. No se muestran los ensayos con cromo debido a que no se observaron halos de inhibición de luminiscencia ni de crecimiento  5. Diameter (average of duplicates) of the growth and luminescence inhibition zone (mm) for different concentration (mg mL -1 ) of cadmium, copper, lead, and zinc ions of bacterial strains with intense luminescence isolated from the golfo de Nicoya, Costa Rica. The diameters of the growth inhibition zones (mm) for Cu and Pb are the same. Strains with dim luminescent (CL6, CL11, CL12, and CL19) did no exhibit growth or luminescent inhibition zones Fig. 5. Diámetro (promedio de las réplicas) de las zonas de inhibición de crecimiento y de luminiscencia (mm) por concentración (mg mL -1 ) de cadmio, cobre, plomo y zinc de cepas bacterianas con luminiscencia intensa aisladas del golfo de Nicoya, Costa Rica. El diámetro de las zonas de inhibición del crecimiento (mm) para Cu y Pb es el mismo. Las cepas con luminiscencia tenue (CL6, CL11, CL12 and CL19) no mostraron zonas de inhibición de crecimiento o luminiscencia showed luminescence inhibition zones for 0.5 and 1 mg mL -1 Cu, while CL13 strain only for 1 mg mL -1 Cu (Fig. 5).
Strains affected by two or more concentrations of the same metal showed a directly proportional relationship between the diameter of the growth and luminescence inhibition zones and the corresponding metal concentration. CL9, CL10, CL13, and CL17 strains showed that diameters of the luminescence inhibition zones vary in the order of Cd > Zn > Cu (Fig. 5).
The mean difference between inhibition zones of growth or luminescence by metal and metal concentration is shown in Table 1. According to the statistical tests, growth inhibition zones by Cd and luminesce inhibition zones by Zn and Cd were significantly higher at 1 mg mL -1 than 0.5 mg mL -1 for each metal. Luminescence inhibition zones for 1 and 0.5 mg mL -1 Cd and 1 mg mL -1 Zn were statistically significantly higher than the corresponding growth inhibition zones. These strains showed higher luminescent inhibition zones (P < 0.05) by Cd than Zn at 0.5 and 1 mg mL -1 . The difference for mean pair-wise comparison for growth inhibition zones by Cd and Zn at 1 mg mL -1 did not show a statistical significance. Table 1. Pair-wise comparison between the diameter of the growth inhibition zone or luminescence inhibition zone caused by different metals at different concentration (mg mL -1 ) levels upon luminous bacteria strains isolated from golfo de Nicoya, Costa Rica Cuadro 1. Comparación por pares entre el diámetro de las zonas de inhibición del crecimiento o zonas de inhibición de luminiscencia causada por distintos metales a diferentes concentraciones (mg mL-1) sobre las cepas de bacterias luminiscentes aisladas del golfo de Nicoya, Costa Rica Notes: *CI = confidence interval; **1 and 0 denote significant and insignificant, respectively, at the 0.05 significance level.

DISCUSSION
This study's culture medium promoted the growth and luminescence of the tested strains; therefore, it was appropriate for the assays. Most research on luminous bacteria use the SWC (Seawater Complete) culture medium (Bagordo et al. 2012;Martini et al. 2013;Jabalameli et al. 2015) or culture media with a similar composition (Efremenko et al. 2014;Drozdov et al. 2015;Urbanczyk et al. 2015). In this study, strains grew over (4 h) in Rev without requiring specialized equipment. It could also be considered a useful technique for the screening of isolated strains and to define the optimal incubation period of each strain in view of evaluating the effects of toxic substances in solid-phase bioluminescence assays.
The luminescence inhibition rate of some bacterial strains has been used in toxicological and ecotoxicological assessments (Menz et al. 2013;Ma et al. 2014). Our results show that some metals at different concentration levels affect the growth and luminescence of luminous bacterial strains to different degrees. Also, we observed that the luminescence of some bacteria was inhibited by a metal ion concentration lower than the minimum growth inhibitory concentration.
The sensitivity of each strain to different concentrations of metal ions is determined by its bioluminescent phenotype and for practical bioassay applications the higher intensity luminescent strains with luminescence inhibition zones afford greater potential as pollution indicators. For example, strain CL17 that is potentially sensitive to cadmium at concentrations lower than 0.1 mg mL -1 .
The intensity of the luminescence (in free and immobilized cells) decreases with increasing metal concentration. Kumar et al. (2015) reported the effect of copper and zinc at 1 mg mL -1 on luminescence sensitivity of 20 the Marine Agar (MA) and luminescence occurred 2 or 4 h later.
The assessment luminescence intensity dynamics of the assayed strains confirms that bacterial luminescence is an indicator of the activity of intracellular metabolic processes. The maximum luminescence intensity in a bacterial culture occurs during the exponential growth phase. Then luminescence dynamics is affected because of the reduction in metabolic processes kinetics (Drozdov et al. 2015). Efremenko et al. (2014) report that the luminescence intensity in free cells of Photobacterium phosphoreum decreases with time.
Differences between assessed strains in terms of luminescence duration and intensity coincide with the findings reported by Efremenko et al. (2014). The authors indicate that duration and intensity significantly differ between species and strains of the Photobacterium genus. In this way, the amount of light produced by luminous bacteria, even belonging to the same genus, makes these microorganisms show different bioluminescent phenotypes even though bioluminescence is regulated by a similar basic genetic system (Jabalameli et al. 2015).
Although the classification of the bioluminescence intensity used in this study is not a quantitative method, it is adequate for the determination of bacterial luminescence dynamics (mainly in solid culture media) Rev Efremenko et al. (2014) showed that luminescence intensity in cells of P. phosphoreum, immobilized in a polyvinyl alcohol cryogel, decreases with increasing metal ion concentration, and further concluded that metal toxicity for these immobilized cells varies in the order Cu +2 > Co +2 > Hg +2 > Zn +2 . Our study reveals the order Cd > Zn > Cu, as well as the absence of luminescence inhibition by Cr +3 and Pb +2 at all the tested concentrations.
Many application tests employing luminous bacteria are known, and their distinctive features depend on the specific characteristics of the bacterial strain used. The isolation of native luminous bacteria allows bioprospecting for strains useful for monitoring environmental toxicity. Moreover, the isolation of strains of microorganisms resistant to metals is significant. The expectation is that microorganisms designated for bioremediation are tolerant of metals (Kumar et al. 2015).

CONCLUSION
Differences in luminescence intensity dynamics between strains, the definition of luminescence inhibition zones, and the determination of growth inhibition zone diameters and luminescence inhibition zone diameters for different metals ions at different concentration levels were determined.
For sensible strains, a range of concentrations of the metal ion lower than the growth inhibition concentration affected the expression of luminescence. Strains showing intense luminescence and defined luminescence inhibition zones were assigned greater application potential as bioindicators for environmental toxicity monitoring. However, more studies are required to determine the minimum growth and luminescence inhibition concentrations for the assayed metals ions and other substances potentially toxic to coastal marine ecosystems in Costa Rica.