Chlorine sensitivity of environmental, distribution system and biofilm coliforms

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Pergamon

WaL Sci. Tech. Vol. 35, No. 11-12, pp. 289-292, 1997. . 1997 IAWQ. Published by Elsevier Science Ud Printed in Greal Britain 0273-1223197 $17'00 + 0'00

PH: S0273-1223(97)00274-6

CHLORINE SENSITIVITY OF ENVIRONMENTAL, DISTRIBUTION SYSTEM AND BIOFILM COLIFORMS D. P. Sartory* and P. Holmes** • Quality and Environmental Services, Severn Trent Water Ltd. Welshpool Road. Shrewsbury Sf3 BB!. UK .. Quality and Environmental Services, Church Wilne WTW, Draycott Road, Long Eaton, NOJO 3AZ, UK

ABSTRACf Coliform bacteria, isolated from treated drinking water supplies, can be derived from a range of sources (e.g. infiltration. breakthrough at the treatment works or from the biofilm established within the pipework). The sensitivity of these bacteria to chlorine may be related to their source and metabolic status. Strains of coliforms were isolated from sewage works effluents, river and reservoir waters as well as from the bulk water and biofilms from distribution systems. These were assayed for sensitivity to free and total chlorine using two assay procedures. For E. coli. the isolates from the distribution system bulk water showed greater resistance to free chlorine than those from sewage effluents and equivalence to those from river waters. For non-E. coli coliforms (mainly strains of Klebsiella. Enterobacter and Citrobacter). those from distribution system biofilms showed the greatest sensitivity to free and total chlorine whilst those from river water had the greatest resistance. @ 1997 IAWQ. Published by Elsevier Science Ltd

KEYWORDS Colifonns; E. coli; chlorine sensitivity; river water; drinking water biofilms. INTRODUCfION Colifonns isolated from treated drinking water supplies may be derived from those surviving breakthrough at the treatment works, failure in integrity of the distribution system or from natural populations growing within an established biofilm on the pipe walls. The ability of thesE. colifonns, once suspended in the flowing bulk water, to survive exposure to the chlorine present may be dependent upon their source and metabolic status. A complex set of factors. both physiological and environmental, may interact to affect bacterial resistance to disinfectants (Olson and Stewart, 1990). Bacteria grown under low nutrient conditions have been shown to be more resistant to chlorine and chloramines than those grown with more nutrients (Wolfe and Olson, 1985; LeChevallier et al., 1988). It is generally accepted that thosE. colifonns associated with biofilms are less susceptible to chlorine than those suspended in the water (Olivieri et al., 1985) due to protection by the biofilm matrix. There are, however, limited data on the relative susceptibility of strains of natural biofilm colifonns, once free from the biofilm matrix, to chlorine, compared with strains isolated from the bulk water or from other environmental waters. This study consisted of a set of simple assessments of the relative sensitivity of several strains of colifonns, isolated from a range of surface water and water supply environments. to total and free chlorine using two assay procedures. 289

290

D. P. SARTORY and P. HOLMES

MATERIALS AND METHODS Colifonns (68 strains) were isolated from sewage effluent. source rivers. a storage reservoir and drinking water distribution systems as well as from the biofllm of exhumed water distribution pipes (Table 1). They were subcultured onto nutrient agar prior to identification and testing for sensitivity to total and free chlorine. The isolates were not subcultured further before testing so as to limit any changes in characteristics due to repeated subculturing (Wolfe and Olson. 1985). Table 1. Identification and source of coliform isolates tested for resistance to chlorine Sewage effluent

E.'iCherichia coli Citrobaclerjreulldii Enterobacter agg/omerans Enlerohacler aml1ige11lls Ellterohacler cloacae Elllerobacler il1termedillm Ellterohacler saka:akii Kfeb.~iella oxytoco Klehsiella plleumot/iae Rhone/Ia (J(pKllilis Kluywra spp Se"aJiajOt/tiCfJla Se"atia liquijaciem Total non-E coli coliforms

2 2

Rivers & reservoirs 9

10

1 I

1 2 5 I I

I 3 3 I I 3 I 3

10

23

2

2

Biofilmfrom distribution

5

2

I

Water from distribution

2 1 1 7

Susceptibility of the isolates to total chlorine was determined using a modified version of the disc assay procedure of Ridgeway and Olson (1982). Overnight nutrient broth cultures of the isolates were spread onto low nutrient agar plates to achieve semi-eonfluent growth. Sterile filter paper discs soaked with 30ml of a 500mg/l solution of sodium hypochlorite were placed on the plates, which were then incubated at 30°C for 24 hours, after which zones of inhibition were measured. Susceptibility to free chlorine was assayed using a membrane filter procedure. Serial dilutions in Ringer's solution were prepared from overnight nutrient broth cultures. Aliquots (l0mL) of these dilutions were filtered using Gelman GN6 membrane filters which were then flooded with a chlorine solution (one of 0.0. 0.25. 0.5. 0.15. 1.0. 1.5 or 2.0mgIL free chlorine prepared from sodium hypochlorite). After a contact time of 2Inin, the filters were rinsed with an excess of a solution of sodium thiosulphate (1.8% w/v). the membranes transferred to plates and incubated at 30°C for 24 hours before counting of the colonies. From the data generated. the chlorine concentration required to cause a 90% reduction in the population of each isolate was calculated.

RESULTS For both total combined and free chlorine exposure, the isolates tested demonstrated a wide range of tolerances to the disinfectant. For the total chlorine exposure, inhibition zone diameters ranged from 1239mm with the variation being principally related to the source of the isolates. Within each source group there was no significant difference between the species' response to the exposure. The non-£. coli coliforms derived from pipe biofllms were markedly more sensitive to total chlorine than those isolated from the bulk water. which in turn were more sensitive than those from sewage effluent and rivers (Table 2). The E. coli isolates from all sources gave equivalent sensitivity reactions.

Chlorine sensitivity

291

Table 2. Relative sensitivity of E. coli and (' .her coliforms to total combined chlorine as determined by disc assay Source ofisolates n Sewage c;ffluent River & reses;voir water Water from disfnoution Biofilm from distribution

2 9 7

E coli isolates Mean zone (mm) 14.5 14.0 16.7

Stddev

n

2.1 0.7 2.9

10 10 23 7

non-E co" isolates Mean zone Std deY (rom)

16.8 15.8 20.0 27.9

2.7 2.9 4.6 6.1

For exposure to free chlorine, the non-E. coli coliform isolates from biofilms were again markedly more sensitive than those from the other three groups (Table 3), being sensitive to half the concentration of free chlorine of those from rivers and reservoirs, whilst those from water in distribution and sewage effluents were intermediate in sensitivity. The E. coli isolates from water in distribution and rivers and reservoirs were nearly twice as resistant to free chlorine as those from sewage effluents. The most resistant isolates from water in distribution were strains of Serratia liquifaciens, Kluyvera spp and Klebsiella pneumoniae and two

E. coli.

Table 3. Relative sensitivity of E. coli and other coliforms to free chlorine as determined by a membrane assay Source of isolates n

E coli isolates Mean chlorine dose (mg/L)* 0.50 0.91 0.93

2 Sewage effluent 9 River & reservoir water 7 Water from distribution Biofilm from distribution *concentrations that result in a 90"/0 reduction in population size

non-E coli isolates Std dev

n

0.0 0.45 0.37

10 8 23 7

Mean chlorine dose (mg/L) 0.73 0.91 0.72 0.45

Std dev 0.32 0.47 0.35 0.27

DISCUSSION It is accepted that bacteria associated with distribution pipe biofilms are more protected than those in the bulk water. This set of simple assessments shows that biofilm coliforms may be more sensitive to chlorine once released from the biofilm matrix. It is known that bacteria acclimatised to little nutrients are more resistant to disinfectants than those grown at higher nutrient levels (Olson and Stewart. 1990). The previous metabolic histories of our isolates may explain the differences, still apparent after a single subculture on a nutrient rich medium, whereby the strains from the bulk water (relatively low nutrients) were more resistant to chlorine whilst those from the biofilm were adapted to a relatively nutrient-rich environment. which resulted in a greater physiological sensitivity to chlorine. Sensitivity to chlorine is related to the nature of the lipopolysaccharide (LPS) component of the cell surface (Calomiris and Scocca, 1994) with the more sensitive species releasing more LPS under chlorination than chlorine-tolerant species. Changes in LPS composition are related to nutrient availability (Stewart and Olson, 1992). Differences in LPS structure of isolates previously nutrient-stressed when suspended in treated water compared with those isolated from biofilms with higher nutrient levels may be a significant factor in the increased sensitivity of the biofilm isolates to disinfection. The survival of biofilm bacteria, once released into the bulk water, is, therefore, probably due more to adherent biofilm matrix than inherent chlorine tolerance.

292

D. P. SARTORY and P. HOLMES

ACKNOWLEDGEMENTS We are indebted to Richard Williams and microbiology staff of Quality and Environmental Services for their technical assistance. This paper is published with the pennission of Severn Trent Water Ltd and the views expressed are those of the authors and do not necessarily reflect those of Severn Trent Water.

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