The role of cypress leaves in promoting growth of coliform organisms in a holding reservoir

Wat. Res. Vol. 28, No. 10, pp. 2147-2151, 1994

Pergamon

0043-1354(94)E0024-Z

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/94 $7.00 + 0.00

THE ROLE OF CYPRESS LEAVES IN PROMOTING GROWTH OF COLIFORM ORGANISMS IN A HOLDING RESERVOIR PETER PASPALIARISl a n d BRIAN HODGSON2. tMelbourne Water, New Farm Road, Werribee, Vic. 3030 and 2Department of Microbiology, University of Melbourne, Parkville, Vic. 3052, Australia

(First received March 1993; accepted in revised form January 1994) Akstraet--Coliforms at levels of up to 4 x 104 per 100 ml were detected in the outlet of an open holding service reservoir for domestic water supplied with chlorinated water that contained no more than 3 coliforms per 100 ml and these were identified as mostly Enterobaeter cloacae and some Citrobacter freundii. These were also the coliforms isolated most frequently from Melbourne Water catchment reservoirs. Growth of these coliform organisms occur within the holding reservoir. Cypress trees (Cupressus macrocarpa) deposited some 6000kg of leaves into the reservoir per annum and this had produced a 3 cm sediment layer of decaying leaves. In the laboratory coliforms and other heterotrophs grew in water containing cypress leaves achieving populations of 3 x 105 per I00 ml and 4 x 10s per ml respectively. Cold water leaf extracts supported the growth of Citrobacter freundii, Enterobacter cloacae, Eseherichia coil, Salmonella derby and uncharacterised heterotrophs present in the reservoir sediments. Hot water extracts, however, contained inhibitors of growth of all species tested except for Enterobacter cloacae. Growth of coliform organisms and other heterotrophs was probably supported by nutrients derived from cypress leaves. In order to preserve the quality of water, reservoirs should be protected from receiving cypress leaves.

Key words--coliforms, Enterobacter cloacae, Citrobacter freundii, domestic water storage, nutrients from cypress leaves

MATERIALS AND METHODS

INTRODUCTION D u r i n g our s t a n d a r d microbiological testing procedures o n potable water supplies, in one open holding service reservoir we o b t a i n e d total coliform counts as high as 4.0 x 104/100 ml in the outlet water. The inlet water to this reservoir is prechlorinated and has a total coliform c o u n t n o t exceeding the 10 per 100 ml r e c o m m e n d e d as the m a x i m u m c o u n t allowable for potable water in Australia (Anon, 1987). Consequently the water was rechlorinated before being t r a n s m i t t e d by the supply system. As up to 400 MI daily pass t h r o u g h this reservoir, this was an added cost, a n d as such we t h o u g h t it necessary to characterise a n d identify the source o f these coliforms. The reservoir is lined o n two sides with large cypress trees (Cupressus macroearpa), which deposit large quantities of cypress leaves into the reservoir where they become waterlogged a n d sink to the bottom. In this p a p e r we identify the coliforms present in the reservoir a n d test various organisms for their ability to grow with aqueous extracts o f cypress leaves or water containing cypress leaves.

*Author to whom all correspondence should be addressed. WR2S/*0--H

The reservoir forms part of the regional distribution system, with the principal function of transporting water in bulk on a daily basis from the seasonal balancing reservoirs to the water supply zones throughout the metropolis. This reservoir has a surface area of 24,000 m 2, a depth of 5.70 m and a capacity of 200 megalitres (MI). At the time of this experiment it was lined on two sides with 36 large cypress trees (Cupressus macrocarpa), which were originally planted on the prevailing wind side of the reservoir to act as a wind break and at that stage each was 20-25 m high. About 200 M1 of water pass through the reservoir per day during winter and 400 MI during summer. There are two inlet mains of 900 and 600 mm diameter, and two outlet mains, of 900 and 1150 mm diameter.

Origin of cultures and bacteriological analysis of water samples and cypress leaves The stock Citrobacter freundii and Enterobacter cloacae cultures used were isolated from the bottom sediment of the reservoir. The E. coli culture was isolated from untreated water taken from Sugarloaf reservoir by the standard method of the Standards Association of Australia (1981). Salmonella derby was an isolate from wastewater and identified by the Microbiological Diagnostic Unit at the University of Melbourne. The numbers of coliforms present in samples was estimated by the standard multi-tube fermentation (MTF) technique of the Standards Association of Australia (10) using the MPN table of the American Public Health Association (1971). For identification, single colonies were transferred by sterile toothpicks from eosin methylene blue (EMB) plates to nutrient agar (NA) plates

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PETER PASPALIARISand BRIANHODGSON

and incubated at 37°C for 24h. The isolates were then identified according to the methods of Cowan and Steel (1974) and the API-20E system, Analtab Products, Plainview, N.Y. The numbers of heterotrophic organisms present was estimated by the standard plate count method of the Standards Association of Australia (1981) at both 37 and 22°C. Cypress leaves were cut from the trees or fallen leaves were collected in empty trays placed under the trees. Cypress leaves were placed in a Waring blender with sterile saline and homogenised for 10 s. Coliform and total counts were then estimated on the supernatant using methods already described. Cypress leaves were dried to constant weight at 80°C for dry weight determinations.

Samples Samples of sediment were collected with an Ekman grab sampler (Welch, 1948). For bacteriological analysis, 10 ml of sediment was added to 90 ml of sterile Ringer's solution and mixed with a super mixer for 1 min. Succeeding tenfold dilutions were made by transferring 10 ml of mixtures to 90 ml of Ringer's solution. Water samples were collected in sterile glass bottles, from taps attached to the inlet and outlet pipes. Collecting bottles contained 0.1 ml of 10% w/v sodium thiosulphate solution per 500 ml of sample to neutralise any chlorine. Prior to sampling, water was run from each tap for 3 min at a rate of approximately 20 litres per min. Samples were processed within 6 h.

Collecting and extracting leaves Fallen cypress leaves were collected in 4 empty trays each with a surface area of 0.36 m E and 30 em deep left on the ground beneath the trees near to the reservoir. In order to calculate the total leaf fall the amount of cypress leaves falling into the trays over a 24 h period was determined on 2 occasions, one in winter the other in summer. Cypress leaves collected in the trays were dried at 100°C for 24 h and then weighed to constant weight. The majority of fallen leaves were green. For the hot water extraction procedure, 30 g of fallen cypress leaves were mixed with 3 litres of sterile distilled water in a 5 litre flask, heated to 80°C, swirled for 20 min and allowed to stand while stirring with a magnetic stirrer and bar for 4 h. For cold water extraction, 7.5 g of fallen cypress leaves were mixed with 500 ml of dechlorinated tap water, swirled for 20 rain and agitated for 4 h at 40°C with a magnetic stirrer and bar. The supernatants were decanted and sterilised by filtration through a 0.45 # membrane filter (Gelman GN-6). In some studies the leaf extract was adjusted to pH 7.0 and/or the ammonia nitrogen content was raised to 5 mg per litre by adding 5% w/v solution of sterile NH4CI. The methods used for determining BOD, total organic carbon (TOC) and pH were those of the American Public Health Association (1980). Flow injection analysis was used to measure ammonium, nitrite, and nitrate nitrogen; orthophosphate and total phosphorus with a Tecator model 5020 (Technicon Instruments Corp., Tarrytown. N.Y.) and the methods described in the "Tecator" application notes. Total Kjeldahl nitrogen (TKN) was determined by procedures taken directly from "Technicon Industrial method No. 37675 w/AU" for continuous flow automatic analyses.

Growth of coliforms with cypress leaves and extract of cypress leaves Coliform cultures to be tested were incubated at 37°C overnight in nutrient broth, centrifuged, the sediment washed 3 times in sterile Ringer's solution, and then resuspended in Ringer's solution. 300 ml of aqueous extract of cypress leaves, or extract adjusted with NaOH to pH 7.0, or water in 500 ml Erlenmeyer flasks were inoculated to give an initial count of between 50-500 coliforms per 100ml. In some experiments 10 g of u.v. sterilised fallen cypress leaves was also added to each incubation flask. In one experiment

a mixture of approximately equal number of Enterobacter cloacae and Citrobacter freundii was inoculated into a suspension of 30 g of cypress leaves in 300 ml of reservoir water to give an initial coliform count of 1 × 10 6 per 100 ml. To obtain a "natural" population of coliforms plus heterotrophs, sediment from a reservoir was mixed with an equal volume of sterile tap water, shaken firmly for 10 min, then the sediment was allowed to settle. For testing the effect of this natural population on the survival of coliforms in the presence of cypress leaves, 10 ml of the supernatant was added to 1 litre of sterile tap water containing 7.5 g of cypress leaves and tested with either no further additions or with an inoculum of either Enterobacter cloacae or Citrobacter freundii. Incubations were at various temperatures. Growth was assessed by measuring the numbers of total coliforms and heterotrophs present as described above over a period of days.

Microbial contamination of water by" wind and fallen leaves Four stainless steel trays each with a surface area of 0.36 m 2 and 30 cm deep were placed one on each side of the reservoir. Each tray was sterilised on site with 80% v/v ethanol:water and rinsed twice with two litres of sterile distilled water. One litre of sterile reservoir water was then added to each tray to act as a trap for bacteria and other matter. After 24 h the contents of each tray were mixed and sampled for heterotrophic organisms growing at 22°C and coliforms by the methods already described. The one tray found to be excessively contaminated with vegetation or bird remains was discarded. In summer when the temperature of the water varied between 14 and 29°C, fresh trays were set up 3 times a week for 6 weeks. In winter when the temperature of the water varied between 5.5 and 10.1°C, each tray was sampled again after 48 h and again after 6 days. In general, during winter, the count on day 6 was used in the calculation. During summer, trays were sampled on 71 occasions after 24 h, whereas during the winter the 72 samples were taken from only 24 trays each sampled at 24 h, 48 h, and 6 days. RESULTS Estimates o f the numbers o f coliforms present in the 900 and 600 m m mains inlet pipes gave coliform counts o f 2 and 3 per 100ml respectively over a 9 m o n t h period o f time. These coliforms were not characterised. In contrast, in the outlet waters estimates o f the coliforms present varied over the same period o f time from 90 to 4 × 104 per 100 ml. The average coliform counts in the 99 and 1150 m m mains outlet pipes over a one year period with weekly sampling was calculated to be 1080 and 700 per 100ml respectively with a 95% confidence limit. Enterobacter cloacae and Citrobacter freundii were the coliforms m o s t frequently detected in the outlets, with Enterobacter cloacae being by far the m o s t d o m i n a n t isolate. The E. coli count in both the inlet or outlet waters was less than or equal to 1 per 100ml in 98% o f samples and was greater than 1 per 100 ml on only 2 occasions. The average heterotrophic count for the 900 and 600 m m mains inlets was 22 and 60 per ml at 37°C and 37 and 123 per ml at 22°C respectively. For the 900 and 1150 m m mains outlet pipes on the other hand the average heterotrophic count was 142 and 100 per ml at 37°C and 600 and 4500 per ml at 22°C

Coliform growth with cypress leaves respectively. These results indicated that the increase in coliform and heterotrophic counts observed between inlet and outlet waters had been derived from a source close to or within the reservoir. In winter the reservoir water temperature ranged from 10 to 12°C while in the summer the range was from 14 to 20°C. From the numbers of heterotrophs and coliforms isolated from the trays of water placed on the side of the reservoir, it was calculated that a total of 3.2 × 1013 heterotrophs and 6.1 x 109 coliforms were introduced each day into the reservoir from the air during summer, while in winter the corresponding numbers each day were respectively l x I012 and 4.6 x 106 organisms. During summer assuming uniform dispersion these values would yield a count in the water of 80 per ml for heterotrophs and 1.5 per 100 mi for coliforms and in the winter 5 per ml and 0.023 per 100 ml respectively which are all significantly less than the counts determined at the outlets. Heterotrophs present on freshly cut cypress leaves were estimated to be 6.0 x 104 and 8.0 x 104 after incubation at 37 or 22°C respectively and coliforms were 0.2 per g of dried cypress leaves. From cypress leaves which fell from the trees the corresponding numbers were 4.1 x 105, 6.6 × 106 and 0.3. It was calculated from the cypress leaves falling into the trays that about 6000 kg of cypress leaves fell into the reservoir per year. This means that the maximum number of coliforms introduced into the reservoir from cypress leaves was approx. 1.8 x l 0 6 coliforms per year which would have resulted in a count of approx. 2 x 10 -7 coliforms per 100ml. Microbiological analysis of the sediment revealed a heterotrophic count at 22°C of 5.0 x 106 organisms per g dry sediment, and at 37°C 2.2 x 106 organisms per g dry sediment. The sediment had a coliform count of 1.3 x 104 coliforms per g dry sediment and E. coli was not detected. A random selection of 100 coliform colonies showed 92 were Enterobacter cloacea, 2 were Citrobacter freundii and the others were not characterised. When 30 g of cypress leaves collected from trees were dispersed in 300 ml of reservoir water and incubated with a mixture of approximately equal numbers of Enterobacter cloacae and Citrobacter freundii after incubation at 22°C the coliform count increased from 1.3 x 106 per 100ml at day 0 to a

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maximum of 1.7 x 109 at day 16. Colonies selected at random from the plate counts at this time were all shown to be Enterobacter cloacae. In the absence of cypress leaves coliform counts decreased from 8 x l05 per 100 ml at day 0 to 2 x l02 per 100 ml at day 8 and were not detected after 12 days' incubation. Cypress leaves therefore contain nutrients that support the growth of Enterobacter cloacae and Citrobacter fruendii. Coliforms, however, constitute less than 1 in 400 of the total population of heterotrophs present in the reservoir sediment, hence we tested the effect of these heterotrophs on the survival of Enterobacter cloacae and Citrobacterfreundii in the presence of cypress leaves. Both coliform counts and total plate counts (Table l) increased rapidly over the first 4 days' incubation and declined slowly over the next 4 days. Other tests showed that in the absence of cypress leaves the population of coliforms decreased to less than 100/100ml after 8 days' incubation, whereas the total plate count remained constant. Five separate colonies selected at random from the EMB plates obtained from the flask containing added Enterobacter cloacae after 4 days were all identified as Enterobacter cloacae whereas only 2 of 5 colonies selected at random from the EMB plates obtained from the flasks containing added Citrobacterfreundii after 4 days were identified as Citrobacter

freundii. The ability of extracts of cypress leaves to support the growth of various species of coliforms and other heterotrophs was dependent on the way in which the extracts were prepared. Cold water extracts supported growth of Citrobacter freundii, Enterobacter cloacae, Escherichia coli and Salmonella derby whereas of the above only Enterobacter cloacae grew with a hot water extract (Table 2). This difference could not be attributed to any differences in the measured parameters (Table 3). Adjusting the pH of the hot extract to 7.0 or adding either ammonium chloride to increase the ammonia-N level to 5 mg/l or cypress leaves also did not allow cultures of Citrobacter freundii or Escherichia coli to grow. Extracts from leaves collected from around other reservoirs that were produced from eucalypts and some deciduous trees did not support the growth of coliforms hence the extracts of cypress leaves are fairly unique in their ability to support the growth of

Table 1. The effect o f a natural population o f heterotrophs on survival o f coliforms at 22°C in tap water containing cypress leaves

Enterobacter cloacae

Citrobacter freundii

Flask Coliforms/100 ml Day Day Day Day Day

0* 2 4 6 8

I 2.1 3.5 9.3 1.5

x × x x ×

101 10 5 l0 5 l0 4 104

Flask Total plate count/ml 2.7 6.5 4.8 1.6 1.1

x x x x x

10 5 10 7 l0 s l0 s 10 s

Coliforms/100 ml 2 4.3 1.7 2.3 9.3

x x x x x

10 3 10 3 l0 5 l0 5 104

Total plate count/ml 3.4 3.5 9.2 6.6 3.9

× x × x x

10 5 107 l0 7 10 7 107

*The 10 ml o f i n o c u l u m o f " n a t u r a l " heterotrophs added to 1 I o f tap water, 7.5 g o f cypress leaves and coliforms, contained 160 coliforms and 3 × l08 heterotrnphs.

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PETER PASPALIARIS a n d BRIAN HODGSON Table 2. Growth at 22°C of pure cultures of heterotrophs on filter stcrilised cypress leaf extracts Total plate counts per 100 ml at 22°C

Citrobacter freundii Days 0 2 3 4 6 7 8 11

Enterobacter cloacae Hot extract

Cold extract

Hot extract

Cold extract

Hot extract

Cold extract

350 35

230 1,4 x 106

50 3.5 x 106

450 5.0 x 106

250 2

150

50

75

<2 <2

1.3 x 108 6.4 × 107

5.0 x 10a 5.0 x 10~

<2 2.4 x 108

<2

<2

7.5 × 107

1.7 x l0 s

9.3 x 108

<2

DISCUSSION

The microbial quality of drinking water is a matter of continuing concern and general interest. Most often this quality is assessed through the detection of indicator organisms such as E. coli and coliforms. Although their detection often indicates that recent faecal contamination has occurred there have been situations described where this was not the case. For example E. coli has been shown to survive and grow in a monomictic reservoir (Gordon & Fliermans, 1978). In the summer of 1983 a persistent coliform bloom was observed in an open finished water reservoir in southern California. The predominant coliform was Enterobacter cloacae and was found to be associated with a concurrent algal bloom and the resident frog population in the reservoir (Olson & Nagy, 1984). In the situation described in this paper where a significant population of coliforms was detected in a holding reservoir this was also not indicative of faecal contamination. Identification of the coliforms showed that they were mainly Enterobacter cloacae and some Citrobacter freundii not Escherichia coli. The coliform and heterotrophic population entering the reservoir via wind action or from the leaves contributes little to the total count in the reservoir. Table 3. Characteristics of hot and cold water extracts of cypress leaves mg/l

TKN TOC Pi Total P Ammonia-N NO2-N NO3-N BOD 25 pH

Salmonelladerby

Cold extract

the coliforms and heterotrophs used in the above experiments.

ParameteP

Escherichia c o i l

Hot extract

Hot

Cold

2.0 48.0 2.1 3,2 0,1 <0.01 <0,01 180 5.5

2,6 41.0 3.4 0.1 <0.01 <0,01 190 5.6

aTKN = total Kjeldahl nitrogen; TOC = total organic carbon; Pi =inorganic phosphorus; Total P = total phosphorus; BOD 25= Biochemical oxygen demand at 25°C, 5 days.

2.3 x 107

<2

2.3 x 108

4.3 x 106

<2

7.5 × 108

6.4 x 104

<2

2.3 x 105

The total coliform count expected from the various sources measured, i.e. inlet, air and present on falling cypress leaves would not exceed 4.5 per 100 ml, still a long way from the 700-1000 observed. The maximum heterotrophic count expected from the various inputs would have been 123 + 80 + 0.4, i.e. 203 per ml, again still some way from the 4500 per ml detected. The coliforms and heterotrophs detected in the outlet mains must therefore have derived from within the reservoir itself. As the heterotroph count at 22°C was always higher than at 37°C it is unlikely that these heterotrophs, which includes the coliforms, were derived from faecal contamination as might occur from birds. They are far more likely to have been derived from soil, vegetation or natural waters. It is well known that trees and leaves contain a great variety of substances extractable with water (Anderson, 1958), and some of these can support the growth of enteric organisms. Klebsiella species and Enterobacter species are capable of growth in aqueous redwood extracts in the presence of indigenous bacteria, whereas most other enteric bacteria cannot (Talbot & Seidler, 1979). Both species have also been found within redwood lumber (Bagley et al., 1978) and staves and water in redwood lined reservoirs (Seidler et al., 1977; Talbot et al., 1979). The predominant carbon source in redwood that can be metabolised by Klebsiella species and Enterobacter species are the cyclitol compounds pinitol, sequoyitol, and myo-inositol (Anderson et al., 1968). In this study no attempt was made to determine the chemical composition of the cypress leaf extract. It can be inferred however, from the high BOD and TOC levels of the extract (Table 3) that large amounts of soluble organic compounds can be extracted from cypress leaves. As it was calculated that 6000 kg of cypress leaves entered the reservoir per year, this must have been the major source of nutrient for the coliforms and heterotrophs. However the type of chemicals extracted vary with the type of extraction employed. Hot water extracts inhibit growth of all the organisms tested in this work except for Enterobacter cloacae. Populations of Enterobacter cloacae of up to l0 s organisms per 100ml were obtained during growth on hot water extracts (Table 2). Similar population levels of Citrobacter freundii, Escherichia coli and Salmonella derby were obtained using a cold

Coliform growth with cypress leaves Table 4. Characterisation of coliform species isolated from 3 Melbourne water catchment reservoirs in 1 year Reservoir 1 Reservoir 2 Reservoir 3 *Yearly total coliforms isolated 1087 440 142 % identified as Enterobacter spp. 62.5 39.1 53.5 Citrobacter spp. 20.4 38.4 31.0 Escherichio coli 13.0 18.6 10.6 Klebsiella '-p. 4. I 3.9 4.9 *Reservoirsare sampled weekly.The numbers represent sum of total coliforms isolated in one year, estimated by the standard MTF technique. All positivetubes of modified mineral salts glutamate were streaked onto cosine methylene blue plates and each a characteristic colony form was identified to species level.

water extract of cypress leaves with a similar BOD content (Tables 2 and 3). The finding that adding cypress leaves to the hot extract did not allow growth of Citrobacter freundii and Escherichia coli also indicates that substances inhibitory to growth were present as Citrobacter freundii did grow in water containing cypress leaves. It might be expected that the degradation of cypress leaves in the reservoir sediment by indigenous organisms would eventually release all nutrients. On visual examination the sediment appears to be composed of decaying cypress leaves. A large population of heterotrophic and coliform organisms are present together with a variety of larger organisms including molluscs, insect larvae, crustacea and diatoms and we assume that the sediment was the source of the coliform organism in the outlet waters. The processes of leaching and microbial colonisation of leaf material in running water has been called conditioning and the process has a significant effect on the types of invertebrates and microorganisms found in the detritus (Triska et al., 1975). Part of the explanation for the dominance of Enterobacter cloacae in the coliforms isolated from the reservoir may be its ability to tolerate these extractable cypress leaf compounds. The original source of the coliforms was probably the inlet water itself. Other work on the range of coliforms present in some of the Melbourne catchment reservoirs confirmed that Enterobacter spp were the commonest isolate (Table 4). Although water from these catchment reservoirs is chlorinated before being transferred to the holding reservoir studied in this work it is unlikely that all organisms were removed. It is likely, therefore, that the coliform counts of 3 per 100ml detected in the inlet water to the holding reservoir were more likely to be due to Enterobacter spp than any other coliform species. The problems of the excessive coliform counts in the reservoir was overcome by removing the cypress

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trees, draining the reservoir, removing the sediment and roofing the reservoir. The rechlorination station has also been removed from the outlet waters. In Australia many farms have cypress trees to act as wind breaks to protect property and many farm dams will accumulate large quantities of cypress leaves. This will have a major impact on the quality of the water in the dam especially if the dam becomes contaminated with animal or bird faeces. Heterotrophs present including perhaps Salmonella spp can multiply with nutrients supplied by the cypress leaves.

REFERENCES

American Public Health Association (1971) Standard Methods for the Examination of Water and Wustewater, 13th Edition. Am. PUbl. Hlth Assoc., Washington, D.C. American Public Health Association (1980) Standard Methods of the Examination of Water and Wastewater, 15th Edition. Am. Publ. Hlth Assoc., Washington, D.C. Anderson A. B. (1958) The composition and structure of wood. J. Chem. Educ. 35, 482-492. Anderson A. B., Riffen R. and Wong A. (1968) Chcmistry of the genus Sequoia. VI on the cyclitols present in heartwood of Sequoia sempervirens. Phytochemistry 7, 1867-1870. Anon (1987) Guidelines for drinking water quality in Australia. NH&MRC and Australian Water Resources Council, Canberra. Bagley S. T., Seidler R. J., Talbot H. W. Jr and Morrow J. E. (1978) Isolation of Klebsielleae from within living wood. AppL envir. Microbiol. 36, 178-185. Cowan S. T. and Steele K. J. (1974) Manual for the Identification of Medical Bacteria, 2nd Edn. Cambridge Univ. Press. Gordon R. A. and Fliermans C. B. (1978) Survival and viability of Escherichia coil in thermally altered reservoirs. Wat. Res. 12, 3443-3452. Olson B. H. and Nagy L. A. (1984) Microbiology of potable water. Adv. Appl. Microbiol. 30, 73-132. Seidler R. J., Morrow J. E. and Bagley S. T. (1977) Klebsielleae in drinking water emanating from redwood tanks. Appl. envir. Microbiol. 33, 893--900. Standards Association of Australia (1981) Microbiological Methods for the Dairy Industry Part 4. Methods for the Examination of Water and Air. Sydney, Australia. Talbot H. W. Jr and Seidler R. J. (1979) Cyclitol utilisation associated with the presence of Klebsisella in botanical environments. Appl. envir. Microbiol. 37, 909-915. Talbot H. W. Jr, Morrow J. E. and Seidler R. J. (1979) Control of coliform bacteria in finished drinking water stored in redwood tanks. J. Am. Wat. Wks Ass. 71, 349-353. Triska F. J., Sedell J. R. and Buckley B. (1975) The processing of conifer and hardwood leaves in two coniferous forest streams. II Biochemical and nutrient changes. Verh. int. Verein. LimnoL 19, 1628-1639. Welch P. S. (1948) Limnological Methods. Blakiston, Philadelphia, Pa.