Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Growth-dependent photoinactivation kinetics of Enterococcus faecalis P.A. Maraccini, D. Wang, J.S. McClary and A.B. Boehm Department of Civil and Environmental Engineering, Environmental and Water Studies, Stanford, CA, USA

Keywords bacteria, batch culture, chemostat, enterococci, growth rate, photoinactivation, sunlight. Correspondence Alexandria Boehm, Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega, Stanford, CA 94305, USA. E-mail: [email protected] 2014/2590: received 15 December 2014, revised 7 February 2015 and accepted 8 February 2015 doi:10.1111/jam.12773

Abstract Aims: To investigate how the growth stage of Enterococcus faecalis affects its photoinactivation in clear water. Methods and Results: Enterococcus faecalis were grown in batch cultures to four different growth stages or grown in chemostats set at four different dilution rates, then harvested and exposed to full spectrum or UVB-blocked simulated sunlight. Experiments were conducted in triplicate in clear water with no added sensitizers. Decay curves were shoulder-log linear and were generally not statistically different in experiments conducted under full spectrum light. Shoulders were longer and first order inactivation rates smaller when experiments were seeded with cells grown to stationary as compared to exponential phase, and for slower growing cells when experiments were done under UVB-blocked light. Chemostat-sourced bacteria generally showed less variability among replicates than batch-sourced cells. Conclusions: The physiological state of cells and the method via which they are being generated may affect the photoinactivation experimental results. Significance and Impact of the Study: Photoinactivation experiments conducted with exponential phase cells may overestimate the photoinactivation kinetics in the environment, particular if UVB-independent mechanisms predominate. Chemostat-sourced cells are likely to provide more consistent experimental results than batch-sourced cells.

Introduction The great economic and social benefits of the coastal environment compel us to keep coastal waters free from microbial pollution that may cause direct or indirect harm to public health (Kildow and Colgan 2005; United States Environmental Protection Agency 2009). The microbial pollutants of greatest concern are human pathogens; however, due to the expense and difficulty of testing waters for all potential human pathogens, waters are tested for faecal indicator bacteria (FIB), including Escherichia coli and enterococci (World Health Organization 2003). FIB concentrations correlate to incidence of recreational waterborne illness when waters are polluted with treated wastewater or urban runoff (Pr€ uss 1998; Haile et al. 1999; Wade et al. 2003; Zmirou et al. 2003; United States Environmental Protection Agency 2009; 1226

Colford et al. 2012). Enterococci concentrations tend to correlate more strongly with illness incidence in marine waters than other indicators (Wade et al. 2003). Therefore, enterococci have become an important microbial pollution assessment tool in the United States and countries around the world (World Health Organization 2003). Recent studies have shown that enterococci concentrations in recreational waters are not constant, but fluctuate over the course of a day (Boehm 2007). Photoinactivation, or inactivation by means of sunlight or radiant energy, naturally attenuates enterococci concentrations in natural waters as documented in both field and laboratory studies (Sinton et al. 1999, 2002; Boehm et al. 2002, 2009). A number of studies have documented photodecay rates of enterococci in the laboratory to obtain rates for use in modelling enterococci concentrations in natural

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

P.A. Maraccini et al.

waters (Sinton et al. 2002; Fisher et al. 2012; Maraccini et al. 2012; Sassoubre et al. 2012). Studies typically seed enterococci grown in the lab into artificial or natural waters and then expose cells to natural or artificial sunlight (Sinton et al. 2002; Fisher et al. 2012; Maraccini et al. 2012). Recent work by Fisher et al. (2012) showed that enterococci cells grown in the lab have distinct photoinactivation rates in natural waters from enterococci cells obtained directly from wastewater. This could be due to the diverse enterococci species in wastewater, which potentially decay at different rates (Maraccini et al. 2012). Alternatively, it could suggest that the physiological states of enterococci seeded from the lab culture and wastewater diverged and resulted in different photoinactivation rates. Previous work with E. coli found that fast-growing E. coli cells were more sensitive to mild heat, UVA light and sunlight stresses than slowgrowing cells (Berney et al. 2006), although another study found that E. coli growth rate did not affect inactivation kinetics with the chemical stressor monochloramine (Berry et al. 2009). Similarly, stationary phase enterococci were more resistant than exponentially growing enterococci to environmental stresses, such as heat, acidity and UV irradiation (Hartke et al. 1998), and the chemical stressor sodium hypochlorite (Laplace et al. 1997; Hartke et al. 1998). A study of the photodynamic decay of Enterobacteriacae and pseudomonads in saline water with an exogenous, synthetic sensitizer Rose Bengal added showed stationary phase cells were more resistant to inactivation that exponentially grown cells (Banks et al. 1985). Growth rate is implicated in the regulation of many stress response functions in bacteria that might explain the observed link between growth rate and inactivation rates. For example, decreased growth rates of E. coli have been linked to increased concentrations of protein Dps that protects DNA from oxidative stress (Azam et al. 1999) and increased levels of guanosine pentophosphate (Wick and Egli 2004) and sigma factor RpoS (Ihssen and Egli 2004; Bucheli-Witschel et al. 2010), both of which are central regulators of general stress response. Increased resistance of stationary phase E. coli cells to oxidants, such as those that may arise during photooxidative stress, relative to E. coli cells in exponential phase is linked to RpoS (Jamieson and Storz 1997; Michan et al. 1999) which controls expression of the sodA gene (Tarassova et al. 2009) that confers resistance to oxidative and osmotic stresses (Hengge-Aronis 2000). In enterococci, growth rate affected the levels of 227 gene transcripts and 56 differentially expressed proteins; however, few genes or proteins showed a growth rate-dependent increase or decrease in expression across three increasing growth rates (Mehmeti et al. 2012).

Enterococcus photoinactivation

Previous work linking inactivation rates to cellular growth rates focused primarily on E. coli with limited work on enterococci. Given the past molecular work showing that growth rate affects expression of enterococci cellular proteins, and that growth rate affects stress response in E. coli, there is reason to suspect that growth rate may affect enterococci photoinactivation rates. In the present study, Enterococcus faecalis was grown in batch cultures and harvested at multiple points along the growth curve, and also grown in and harvested from chemostats operated at different dilution rates. We suspended the harvested cells in buffered, clear water with no added sensitizers, exposed them to simulated sunlight, both full spectrum and UVB-blocked light, measured the inactivation kinetics, and compared inactivation kinetics across different treatments. The goal of this research is to determine the effect of growth stage on the photoinactivation of batch culture-sourced Ent. faecalis and the effect of growth rate on the photoinactivation of chemostatsourced Ent. faecalis in clear water with no added sensitizers. We controlled for the potentially confounding factor of variability of the bacterial seeding material by conducting experiments in triplicate, with each replicate prepared with an identically grown, yet distinct bacterial seed. Because enterococci are important faecal indicator organisms in microbial pollution assessment, a better understanding of their physiologically dependent photoinactivation kinetics will aid in understanding their fate in the environment. The results may also extend to other bacterial species. Materials and methods Organism and experimental setup Tryptic soy broth (TSB; BD Difco, Sparks, MD) was used as growth medium and made following manufacturer’s direction using deionized water. TSB was subsequently filter-sterilized using a Millipore (Billerica, MA) Steritop filter unit (PES membrane, 0
22 lm pore size) (Berney et al. 2006). To prevent foaming of the growth medium when used for chemostat experiments, the TSB was diluted to 25% strength and autoclaved at 121°C for 15 min prior to filtration. Enterococcus faecalis V583 was generated from a concentrated stock stored at 80°C, inoculated into TSB, incubated for 8 h at 37°C on a shaker at 200 rev min1, mixed with 20% glycerol, and thereafter stored at 20°C as a single stock. This single 20°C stock was used as the initial inoculum for all experiments to minimize variance due to starting bacterial population. For experiments investigating the differences in photoinactivation kinetics of Ent. faecalis grown in batch

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

1227

Enterococcus photoinactivation

P.A. Maraccini et al.

cultures for different lengths of times, a loop from the 20°C stock was inoculated into TSB and incubated at 37°C on a shaker at 200 rev min1. After 12 h of incubation, the culture was diluted into four 50 ml centrifuge tubes (polyethylene, Corning Life Sciences, Tewksbury, MA) containing 25 ml TSB to reach an optical density of 0
002. The optical density was measured with a Hach DR 2800 Spectrophotometer (Loveland, CO) using 670 nm wavelength light (Koch 1994). The four replicates of loosely capped centrifuge tubes were again placed at 37°C on a shaker at 200 rev min1 and incubated for four different durations (4, 8, 12 and 24 h; Table 1), with each tube serving to generate the experimental seed for only one of the four time points. This entire procedure, from taking the loop of frozen stock to the four replicate centrifuge tubes incubated for four different durations, was repeated six times to generate distinct seed stock for three replicates under full spectrum and three replicates under UVB-blocked radiation for each growth stage condition (Figure S1, Table 1). The 4, 8, 12 and 24 h old cultures are referred to as mid-exponential (ME), early stationary (ES), mid-stationary (MS) and late stationary (LS), respectively. For experiments investigating the differences in photoinactivation kinetics of Ent. faecalis grown in

chemostats at different dilution (growth) rates, a loop from the 20°C stock was inoculated into TSB and incubated at 37°C on a shaker at 200 rev min1. After 6 h of incubation, the culture was diluted into four chemostats (borosilicate, Adams and Chittendens, Berkeley, CA) of varying volumes to reach an optical density of 0
002. The chemostats were immersed in a 37°C water bath. The volumes of the chemostats (20, 15, 10 and 5 ml) and the flow rate of the influent TSB medium (0
062 ml min1) were controlled to obtain the desired growth/dilution rates (3
1 9 103, 4
2 9 103, 6
2 9 103 and 12 9 103 min1; Table 2). The chemostat dilution rates spanned the range of the batch culture specific growth rates during mid-exponential to early stationary phase, and approximately matched the dilution rates of past studies that attempted to document growth dependent decay kinetics of various bacteria (Gilbert and Brown 1978; Berg et al. 1982; Harakeh et al. 1985; Humpheson et al. 1998; Berney et al. 2006; Berry et al. 2009). After the OD (measured at 670 nm) stabilized (approx. 8 h), Ent. faecalis cells were harvested from the chemostats to serve as the experimental seed. Chemostats were run up to 48 h to complete all planned experiments. Identical chemostats filled with TSB but unseeded with bacteria were run in parallel with the experimental chemostats to

Table 1 Batch culture experiment growth details (replicate, growth stage, time of growth, specific growth rate, initial Enterococcus faecalis concentration in reactor), shoulder-log linear fitting information (k and S values  95% confidence interval, Pearson’s v2 test values) and T99
9%

Replicate

Growth stage

Time of growth (h)

Specific growth rate, l (min1)

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 5 5 5 6 6 6

ME ES MS LS ME ES MS LS ME ES MS LS ME ES LS ME ES LS ME ES LS

4
1 8
1 12
4 24
1 4
0 8
1 12
3 24
3 4
1 8
1 12
0 24
3 4
0 8
1 24
1 4
1 8
0 24
0 4
1 8
0 24
1

1
9 5
7 2
7 3
4 1
7 1
7 2
6 4
2 2
2 1
7 2
0 1
2 1
9 1
2 1
4 2
0 9
3 9
6 2
0 1
2 1
3

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

102 104 107 1014 102 104 107 1015 102 104 107 1014 102 104 1015 102 105 1016 102 104 1014

Initial conc. in reactor, log10 (CFU ml1)

UVB cut-off filter used?

6
4 7
1 7
1 7
2 6
8 7
4 7
3 7
4 6
2 7
2 7
2 7
2 6
7 7
2 7
3 6
4 7
3 7
4 6
4 7
0 7
0

No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes

Pearson’s v2 test k (min1) 0
64 0
44 0
50 0
49 0
60 0
60 0
57 0
46 0
45 0
60 0
54 0
43 0
077 0
075 0
071 0
10 0
11 0
091 0
075 0
070 0
057

                    

0
21 0
14 0
06 0
03 0
16 0
16 0
11 0
08 0
08 0
11 0
10 0
05 0
051 0
011 0
013 0
00 0
02 0
013 0
012 0
005 0
014

S (min)

v2

n

P-value

T99
9% (min)

                    

1
7 2
9 0
3 0
1 1
1 1
5 0
9 0
7 0
6 0
7 0
9 0
1 0
6 0
6 1
2 0
0 0
8 0
7 1
2 0
1 2
0

6 7 7 7 6 7 7 7 6 7 7 7 4 7 7 3 7 7 7 6 7

0
89 0
82 1
00 1
00 0
95 0
96 0
99 0
99 0
99 0
99 0
99 1
00 0
89 1
00 0
97 1
00 0
99 0
99 0
98 1
00 0
92

46 54 63 51 50 61 65 59 44 63 66 66 230 520 460 170 510 500 110 300 410

35 38 49 36 39 50 53 44 29 52 53 50 140 420 360 98 450 420 14 210 290

9 11 4 2 7 7 5 5 6 5 5 3 110 40 50 0 30 30 34 20 70

ME, Mid Exponential Phase; ES, Early Stationary Phase; MS, Mid Stationary Phase; LS, Late Stationary Phase.

1228

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

P.A. Maraccini et al.

Enterococcus photoinactivation

act as controls and ensure there was no contamination due to chemostat operation. This entire procedure, starting with the inoculation of frozen stock to the operation of stable chemostats, was repeated six times to generate distinct seed stock for three replicates under full spectrum and three replicates under UVB-blocked radiation for each growth rate condition (Figure S2, Table 2). As the dilution rate varied slightly between replicate experiments (by at most 0
1%), they are referred to as dilution groups A, B, C and D corresponding to cells grown in chemostats with dilution rates of 3
1 9 103, 4
2 9 103, 6
2 9 103 and 12 9 103 min1, respectively. The bacterial seed was washed before use in the inactivation experiments. One ml was withdrawn from batch cultures or chemostats, pelleted by centrifugation (9600 g for 10 min), decanted and re-suspended in autoclaved carbonate buffered saline (CBS, 1 mmol l 9 NaHCO3, pH = 7
64, 20–25°C, Fisher Scientific, Fair Lawn, NJ; all salts reagent grade or better). This was repeated three times, and then diluted into a CBS solution at a volumetric ratio of 1 : 99 to a final volume of 50 ml in sterile 100 ml glass beakers wrapped with black electrical tape.

Beakers were placed in a recirculating water bath to maintain their temperature at 15°C within a solar simulator (Altas Suntest XLS+; Chicago, IL) equipped with a window glass filter (Cat. 56052372; Atlas MTS, Chicago, IL). A second UVB cut-off filter (FSQ-WG320, Newport, Franklin, MA) was placed atop the beaker for select experiments to block UVB irradiation, which mimics the solar spectrum of deeper waters where UVB wavelengths do not penetrate. The solar simulator intensity was set to 400 W m2. The light spectra of the solar simulator and natural sunlight (Stanford, CA 1130 h on 9 July 2014) were measured using a spectroradiometer (ILT950, International Light Technologies, Peabody, MA), which demonstrated that the solar simulator had similar intensities to natural sunlight over the UV range (Figure S3). Solutions were allowed to acclimate for 10 min in the dark prior to initiation of the light irradiation. Replicate beakers covered with foil were placed in the same recirculating water bath to serve as dark controls. All experiments were replicated three times with either distinct batch- or new chemostat-generated bacterial seeds (Figures S1 and S2, Tables 1 and 2). In total, 45 experiments were conducted: eight experiments in

Table 2 Chemostat experiment growth details (replicate, dilution group, dilution rate, initial Enterococcus faecalis concentration in reactor), shoulder-log linear fitting information (k and S values  95% confidence interval, Pearson’s v2 test values) and T99
9%

Replicate

Dilution group*

Dilution rate, (min1)

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6

A B C D A B C D A B C D A B C D A B C D A B C D

3
1 4
1 6
2 1
2 3
1 4
1 6
2 1
2 3
1 4
2 6
2 1
2 3
2 4
3 6
4 1
3 3
1 4
1 6
2 1
2 3
0 4
0 6
0 1
2

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

103 103 103 102 103 103 103 102 103 103 103 102 103 103 103 102 103 103 103 102 103 103 103 102

Pearson’s v2 test

Initial conc. in reactor, log10 (CFU ml1)

UVB cut-off filter used?

6
9 6
9 6
9 6
8 6
4 6
8 6
8 6
6 5
9 6
7 6
8 6
7 6
3 6
4 7
0 6
8 6
1 6
3 6
8 6
6 6
6 6
3 6
9 7
2

No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

k (min1) 0
46 0
52 0
55 0
47 0
46 0
38 0
51 0
51 0
42 0
41 0
36 0
43 0
024 0
033 0
088 0
084 0
025 0
014 0
049 0
088 0
013 0
012 0
052 0
032

                       

0
25 0
11 0
09 0
08 0
10 0
07 0
11 0
18 0
06 0
09 0
03 0
12 0
007 0
012 0
015 0
017 0
017 0
010 0
019 0
024 0
006 0
004 0
021 0
016

S (min)

v2

n

P-value

T99
9% (min)

                       

2
5 0
7 1
3 1
0 1
1 0
8 1
7 1
3 0
2 1
0 0
2 1
8 0
1 0
4 1
1 1
6 0
3 0
1 0
8 1
3 0
1 0
1 0
3 2
3

6 7 7 7 7 7 7 6 7 7 7 7 7 7 7 7 5 5 7 7 7 7 7 7

0
78 0
99 0
97 0
99 0
98 0
99 0
95 0
94 1
00 0
99 1
00 0
94 1
00 1
00 0
98 0
95 0
99 1
00 0
99 0
97 1
00 1
00 1
00 0
89

68 65 54 59 59 57 55 53 71 58 55 60 620 490 310 290 520 830 460 400 810 710 530 380

53 51 41 44 44 39 42 40 54 41 36 44 330 280 230 210 250 330 310 320 270 110 390 160

14 6 5 6 7 7 7 8 4 7 4 9 40 60 30 40 130 110 60 40 80 110 40 120

*Experiments in the same Dilution Group (A, B, C or D) have approximately the same dilution rate.

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

1229

Enterococcus photoinactivation

P.A. Maraccini et al.

triplicate under full spectrum irradiation and seven experiments in triplicate under irradiation with a UVB cut-off filter. A batch culture was not harvested at 12 h for the UVB cut-off filter experiments due to required length of the experiments and the resultant lack of space in the experimental setup (Figure S1). Organism enumeration during photoinactivation Upon exposure to the solar simulator light source, 0
5 ml samples were withdrawn from the beakers over a time course (every 15 min for 90 min for full spectrum irradiation experiments, every 60–120 min over 9–12 h for experiments with the UVB cut-off filter). Samples were serially diluted with autoclaved CBS. Enterococcus faecalis were enumerated by spread plating appropriate dilutions in duplicate on tryptic soy agar (TSA, BD Difco). TSA plates were incubated at 37°C, and colonies were counted after 48 h. Concentrations were calculated using counts from all plates with between 10 and 400 colonies after accounting for the dilution and volume applied to the agar. Effect of initial starting Enterococcus faecalis concentration The initial starting Ent. faecalis concentration in the experiments varied between 7
9 9 105 and 2
5 9 107 CFU ml1 (or 5
9 and 7
4 log10(CFU ml1)) (Table 1 and 2). High bacterial concentrations in experimental solutions could act to shield bacteria from incident photons (Ormeci and Linden 2002); therefore, we sought to explore if the different initial starting Ent. faecalis concentrations would affect fluence in the experimental beakers. We prepared two solutions of a chemical actinometer solution and then inoculated one with a high concentration of Ent. faecalis. The two solutions were exposed to full spectrum and UVB-blocked simulated sunlight in the solar simulator side-by-side and the actinometer decay rates were compared to determine if they differed. P-nitroanisole (PNA) with the addition of pyridine (PYR) acted as the actinometer (Dulin and Mill 1982). This actinometer is sensitive to light between wavelengths of 300 and 370 nm (Dulin and Mill 1982), which overlaps the UVB (280–315 nm) and UVA (315–400 nm) light spectra. The actinometer solution was prepared according to Leifer (1988). Briefly, 0
0153 g PNA (99 + %; Acros Organics, Fair Lawn, NJ) was dissolved in 10 ml of acetonitrile, of which 1 ml was added to 1 litre of 0
1 lm filtered Nano-pure water. A volume of 100
75 ll PYR (anhydrous, 99
8%; Sigma-Aldrich, Saint Louis, MO) was added to the mixture. An Ent. faecalis inoculate prepared identically to those used for the pho1230

toinactivation experiments was pipetted into the PNA/ PYR solution. The resulting Ent. faecalis concentration was 2
4 9 107 CFU ml1 (or 7
4 log10(CFU ml1)), the highest initial starting concentrations of all photoinactivation experiments (Table 1 and 2). Samples were withdrawn and filtered through a 0
2 lm pore size nylon filter (National Scientific, Rockwood, TN) to remove bacterial particulates. Actinometer samples were stored in the dark in the 4°C room and processed within 24 h on an Agilent 1200 series HPLC (Santa Clara, CA) with an Agilent Eclipse XDB-C18 column. Calculation of specific growth rates For each batch culture, optical density measurements were taken every 30 min until the cultures reached early stationary phase (8–10 h) and then taken every 1–6 h thereafter for up to 24 h. Each optical density curve was fitted with a generalized logistic function: ODðtÞ ¼

K  OD0 ert K þ OD0 ðert  1Þ

ð1Þ

where t is time (min), OD(t) is the optical density at time t (), OD0 is the optical density at time 0 (), K is the carrying capacity () and r is growth rate (min1) (Jolicoeurt and Pontier 1989; Goudar et al. 2005). The fitted values K and r are non-negative and determined using a least-squares fitting in the software IGOR PRO (WaveMetrics Inc., Lake Oswego, OR). The change in the OD was assumed to mirror the change in the concentration of the bacteria batch culture (Koch 1994). Therefore, the specific growth rate, l, is calculated from the logistic model for each individual batch culture.   dOD OD ¼ r  OD 1  ð2Þ lðtÞ ¼ dt K For a chemostat at steady state, the specific growth rate is equal to the dilution rate D (min1) (Novick and Szilard 1950): l¼D¼

TSB Flow Rate Chemostat Volume

ð3Þ

Data analysis A shoulder-log linear model was used to fit the inactivation data from the 45 experiments (Geeraerd et al. 2005):   CðtÞ ekS ¼ ekt ð4Þ C0 1 þ ðekS  1Þekt where t is time (min), C (CFU ml1) is the measured concentration at time t, C0 is the measured concentration at time 0, S (min) is the shoulder or lag time over

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

P.A. Maraccini et al.

Enterococcus photoinactivation

which there is minimal inactivation of ENT, and k (min1) is the rate constant for the log linear portion of the inactivation curve after completion of the lag time. The fitted values k and S are non-negative and determined using a least-squares fitting in the software IGOR PRO (WaveMetrics Inc., Lake Oswego, OR). The model is empirical and does not consider the possible mechanisms of bacterial inactivation (Geeraerd et al. 2005). The time to 99
9% inactivation, T99
9% (min), was calculated for each experiment based on the fitted k and S values from Eqn 5. T99
9% 

lnð1000Þ þS k

ð5Þ

A v2 value was generated for each shoulder-log linear model fitting (Eqn 4) and the Pearson’s v2 test for goodness of fit was used to determine the P-value of each fitting, with the assumed null hypothesis being that the shoulder-log linear model predicted the experimental data. The larger the P-value, the better the data fits the model. Analysis of variance (ANOVA) was performed to examine if the differences in the fitted values k and S between experiments could be attributed to diverse specific growth conditions of the seeded bacteria. For experiments investigating the differences in fitted values for Ent. faecalis grown in batch cultures to different growth stages, the two factors were ‘replicate’ and ‘growth stage’ (Table 1), where replicate refers to the three replicates completed with distinct seed material generated in unique batch cultures and growth stage refers to the cells being in mid-exponential (ME), early stationary (ES), mid-stationary (MS) or late stationary (LS) stage. For experiments investigating the differences in photoinactivation kinetics between Ent. faecalis grown in chemostats operated at different dilution rates, the two factors were ‘replicate’ and ‘dilution rate’ (Table 2), where replicate refers to the three replicates completed with seed material generated in distinct chemostats and dilution rate refers to 3
1 9 103 (A), 4
2 9 103 (B), 6
2 9 103 (C), or 12 9 103 min1 (D). Post hoc Tukey’s tests were performed to determine which growth stages or dilution rates led to significantly different k or S fitted values. Pvalues < 0
05 were considered significantly different. Results Growth curves Specific growth rates of bacteria seeds used in the experiments were calculated and are presented in Tables 1 and 2. They vary from 9
6 9 1016 to 0
022 min1 for the batch-sourced bacterial seeds and 0
0030–0
013 min1 for

the chemostat-sourced bacterial seeds. The growth curve of each batch culture is shown in Figure S4. Photoinactivation of Enterococcus faecalis A shoulder during which change in Ent. faecalis concentration was minimal was observed in all experiments, after which decay appeared to be first order (Fig. 1). Inactivation curves were fit using a shoulder-log linear model, with the k and S fitted values provided in Tables 1 and 2 and graphed in Figures 2 and 3. The Pearson’s v2 test values for all fitted curves were above 0
05, which verified that the shoulder-log linear model predicted the experimental data well. For the full spectrum irradiation experiments, k ranged from 0
36 to 0
64 min1, S ranged from 29 to 54 min and T99
9% from 44 to 71 min. For experiments conducted using the UVB cut-off filter, inactivation was much slower with k ranging from 0
012 to 0
11 min1, S from 14 to 450 min and T99
9% from 110 to 830 min. The dark controls showed minimal decay and the slopes did not differ significantly from zero as analysed by linear regression (P > 0
05) for all experiments except the dark control for Batch Replicate 6 – Growth Stage ES (Figure S5). The rate constant of that dark control, following 1st order decay kinetics, was 0
00031  0
00016 min1, which is orders of magnitude below the calculated rate constant for corresponding photoinactivation experiment (k = 0
070  0
005 min1). Therefore, the dark controls verified that non-solar irradiation factors negligibly impacted the inactivation results. To test whether the presence of relatively high initial starting concentrations of Ent. faecalis affected UV-range fluence in the reactors, the decay rates of p-nitroanisole in the actinometer solution with and without Ent. faecalis were compared using multiple linear regression (Neter et al. 1990). The slopes were not significantly different (P > 0
05, data not shown); therefore, assuming the bacterial particulates have a equally negligible effect over the entire UV and visible light range as they did over the wavelength range of the actinometer (300–370 nm), then the varying initial starting concentrations of Ent. faecalis in the beaker did not substantially affect the overall fluence experienced by the microbes. Batch culture-sourced Enterococcus faecalis inactivation trends Growth stage significantly affected the length of the shoulder, S, for batch-sourced Ent. faecalis in experiments irradiated with full spectrum light and light passing through the UVB cut-off filter (P < 0
05, Table 3). The shoulder was significantly shorter for cells harvested at

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

1231

Enterococcus photoinactivation

P.A. Maraccini et al.

Enterococcus faecalis concentration as In (C/C0)

E. faecalis from batch culture exposed to full spectrum light

E. faecalis from batch culture exposed to UVB blocked filter

0

0

–4

–4

–8

–8

–12

–12 0

20

40

60

80

100

0

E. faecalis from chemostat exposed to full spectrum light 0

–4

–4

–8

–8

Inactivation constant k (1/min) Inactivation constant k (1/min)

600

80

–12 0

20

40 60 Time (min)

80

100

0

E. faecalis from batch cultures exposed to full spectrum light

200

400 600 Time (min)

80

E. faecalis from batch cultures exposed to UVB blocked light

0·8

0·12

0·6

0·08

0·4 0·04

0·2 0·0

Batch 1

Batch 2

Batch 3

0·00

E. faecalis from chemostats exposed to full spectrum light

Batch 4

Batch 5

Batch 6

E. faecalis from chemostats exposed to UVB blocked light

0·8

0·12

0·6

0·08

0·4 0·04

0·2

0·00

0·0 Chemostat Chemostat Chemostat 1 2 3

Chemostat Chemostat Chemostat 4 5 6

mid-exponential phase as compared to mid-stationary phase under full spectrum light, and as compared to cells harvested at early stationary and late stationary phases under UVB blocked light (P < 0
05, Table 3, Fig. 3). Growth stage was not significant in the ANOVA for k from full spectrum and UVB cut-off filter experiments (P > 0
05, Table 3, Fig. 2). Replicate was a significant factor in the ANOVA for both k and S (P < 0
05, Table 3, Figs 2 and 3) in experiments conducted with the UVB cut-off filter. Replicate was not a significant factor for k and S derived for experiments conducted under full spectrum light. 1232

400

E. faecalis from chemostat exposed to UVB blocked filter

0

–12

200

Figure 1 Photoinactivation curves of Enterococcus faecalis V583 grown in a batch culture or chemostat under full spectrum irradiation or a UVB cut-off filter. In the top two panels, Ent. faecalis V583 was grown in a batch culture to the mid-exponential (●), early-stationary (□), mid-stationary (▼) and late-stationary (M) phases. In the bottom two panels, Ent. faecalis V583 was grown in a chemostat at dilution rates of 0
0031 min1 (●), 0
0042 min1 (□), 0
0062 min1 (▼) and 0
012 min1 (M). Replicate photoinactivation experiments for each batch culture growth stage and chemostat dilution group were performed three times.

Figure 2 Inactivation constants, k, and their 95% confidence intervals from shoulder-log linear fitting of photoinactivation curves of Enterococcus faecalis V583 grown in batch cultures or chemostats. In the top two panels, Ent. faecalis V583 was grown in a batch culture to the mid-exponential (■), earlystationary ( ), mid-stationary ( ) and latestationary ( ) phases. In the bottom two panels, Ent. faecalis V583 was grown in a chemostat at dilution rates of 0
0031 min1 (■), 0
0042 min1 ( ), 0
0062 min1 ( ) and 0
012 min1 ( ).

Chemostat-sourced Enterococcus faecalis inactivation trends Growth rate significantly affected k when chemostatsourced Ent. faecalis were irradiated with light under the UVB cut-off filter (P < 0
05, Table 3), but did not affect k when bacteria were irradiated under full spectrum light. Enterococcus faecalis growing with a specific growth rate of approx. 0
012 min1 (group D) tended to have a larger k than cells with lower specific growth rates (groups A and B) under the UVB cut-off filter (Table 3, Fig. 2). Growth rate did not significantly affect S. Replicate was

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

P.A. Maraccini et al.

Enterococcus photoinactivation

500

60

400

40

300

100

0

0 Batch 2

Batch 3

E. faecalis from chemostats exposed to full spectrum light

Batch 4 500

60

400

40

300

Batch 5

Batch 6

E. faecalis from chemostats exposed to UVB Blocked light

200

20 0

E. faecalis from batch cultures exposed to UVB Blocked light

200

20

Batch 1

Shoulder length S (min)

Figure 3 Shoulder length, S, and their 95% confidence intervals from shoulder-log linear fitting of photoinactivation curves of Enterococcus faecalis V583 grown in batch cultures or chemostats. In the top two panels, Ent. faecalis V583 was grown in a batch culture to the mid-exponential (■), earlystationary ( ), mid-stationary ( ) and latestationary ( ) phases. In the bottom two panels, Ent. faecalis V583 was grown in a chemostat at dilution rates of 0
0031 min1 (■), 0
0042 min1 ( ), 0
0062 min1 ( ) and 0
012 min1 ( ).

Shoulder length S (min)

E. faecalis from batch cultures exposed to full spectrum light

100 Chemostat Chemostat Chemostat 1 2 3

not a significant factor for k or S under full spectrum nor under UVB cut-off (P > 0
05, Table 3). Discussion Experiments were conducted under either full spectrum or UVB-blocked simulated sunlight to document the photoinactivation of Ent. faecalis in clear water with no added sensitizers to mimic conditions in clear natural waters. Replicate experiments conducted with bacteria grown at different rates or to different growth stages all showed shoulder-log-linear decay, yet with diverse inactivation parameters. We sought to investigate whether the diverse inactivation parameters could be attributed to diverse growth rates or stages of the starting cultures, or were purely a stochastic artifact attributable to the variation in bacteria in the experimental seed. Growth stage or rate was found to significantly affect the length of the shoulder S and the first order inactivation rate constant k under particular conditions. Specifically, growth stage of cells originally sourced from batch cultures significantly affected the shoulder length in experiments conducted under both full spectrum and UVB blocked light. For chemostat-sourced cells, specific growth rate affected the k value in UVB blocked light. Given the apparent decreased affect of growth rate/stage in the full spectrum experiments relative to the UVBblocked experiments, it appears that inactivation of cells via UVB-dependent mechanisms is less sensitive to the cells’ growth stage or rate. In clear water, UVB damages cells by inducing DNA damage via pyrimidine dimerization (Malloy et al. 1997), which may be less dependent on growth conditions as opposed to other inactivation mechanisms occurring in clear water that do

0

Chemostat Chemostat Chemostat 4 5 6

not depend on UVB irradiation. Such mechanisms may be endogenous indirect mechanisms that involve generation of reactive species, such as singlet oxygen, within the cell (Sassoubre et al. 2012). Note that it was beyond the scope of this study to investigate which molecules in the enterococcal cell are capable of acting as sensitizers; however, we have included absorbance spectra of the aqueous matrix, and the aqueous matrix with cells at the start and end of an inactivation experiment in the supporting materials (Figure S6). When growth stage or rate was a significant factor affecting photoinactivation, slower growing or stationary phase cells exhibited longer shoulders and smaller k values than faster growing cells. Physiological changes within the cell that occur naturally during growth may explain these differences in photoinactivation kinetics. A stress response may arise due to the limitation of nutrients at early stationary phase (Hartke et al. 1998). The conditions may trigger a more robust stress response in the organisms, allowing the cells to persist longer under photo-oxidative stress upon exposure to solar irradiation (Hartke et al. 1998; Ihssen and Egli 2004; Berney et al. 2006). The growth dependent photoinactivation kinetics of Ent. faecalis in this study agree with similar work done with E. coli K12, in that both slow growing E. coli and slow growing Ent. faecalis were less sensitive to sunlight stress (Berney et al. 2006). Banks et al. (1985) also showed that stationary-phase cells of a variety of bacterial organisms were more resistant to photodynamic inactivation via the synthetic sensitizer Rose Bengal than exponential-phase cells. Our work was limited to a laboratory strain of Ent. faecalis, which may respond differently to changing growth conditions as compared to environmentally derived enterococci (Fisher et al. 2012). In addition,

Journal of Applied Microbiology 118, 1226--1237 © 2015 The Society for Applied Microbiology

1233

1234

A