- Email: [email protected]
Ecology of selected bacteria in a small intermittent sewage pond
Water Research Pergamon Press 1970. Vol. 4, pp. 341-351. Printed in Great Britain
ECOLOGY OF SELECTED BACTERIA IN A SMALL INTERMITTENT SEWAGE POND F. J. POST Department of Bacteriology and Public Health, Utah State University, Logan, Utah 84321, U.S.A.
(Received 10 November 1969) Abstraet--A small 24 hr retention time sewage oxidation pond in Logan, Utah has been studied to determine if any relationship exists between the population levels of certain bacteria and selected physical factors of the environment. A high degree of relationship has been found between outlet water temperature and certain groups of special fecal indicators. Fecal enterococci (as measured by the K F method) proved to be the most sensitive to environmental conditions with a correlation coefficient to temperature cubed of --0.857. Fecal Escherichia coli was also highly sensitive with a correlation coefficient of --0.752. This last group proved to be more sensitive than the coliform group as a whole (-0.608). INTRODUCTION
SEWAGEstabilization ponds have become a popular method for the disposal of municipal and industrial wastes in recent years. Concern over bacteria and viruses of public health importance has lead to numerous studies on the disappearance or survival of indicator bacteria and pathogens in these ponds (MALINA and YOUSEF 1964; PUBLIC HEALTH SERVICE, 1960). Few studies have been undertaken relating these changes to the overall bacterial ecology of the ponds. Factors that are related to the disappearance, growth or succession of the bacteria which directly affect the operation of the ponds and the bacteria themselves have been little studied. Reports of the last few years indicate an increasing interest in this direction, however (BARNEKOW and DAVIS, 1967; COOPER,1967; HALVORSONet al., 1968; HORNIGet al., 1964).
N /
p
t
IrricJation
I
~s~ . . . . . °.
/
[ Outlet
t,t-'-X,
(winter) ~
I
To
"-'
/o
hatchery ,,,
w
Loqan City
-
Loqan
S..~ter
River ,2 Marshy Sprino o Well
IrrkJation
FIG. 1. Map of sewage-irrigation pond, Logan, Utah (not to scale). 341
342
F . J . POST
In 1965 L o g a n , U t a h a n n o u n c e d the i n t e n t i o n o f c o n s t r u c t i n g a series o f waste s t a b i l i z a t i o n p o n d s to serve a c o m m u n i t y o f 25,000 to 30,000 people. This p r o v i d e d a n excellent o p p o r t u n i t y to s t u d y certain aspects o f the b a c t e r i a l ecology o f these p o n d s b e g i n n i n g with their inception. P r i o r to the c o m p l e t i o n o f these p o n d s ( N o v e m b e r 1967) L o g a n h a d been d i s c h a r g i n g sewage into two surface streams (FIG. 1) at the city limits w h i c h later flowed t o g e t h e r a n d thence into the L o g a n River, a t r i b u t a r y o f the Bear River flowing into the G r e a t Salt Lake. D u r i n g the s u m m e r m o n t h s ( m i d - M a y to m i d - O c t ) the sewage s t r e a m was d a m m e d i m p o u n d i n g a b o u t 15 million gallons o f w a t e r for a p p r o x i m a t e l y 20-24 h r (TABLES 1, 2). This p o n d served as a sewage s t a b i l i z a t i o n p o n d d u r i n g the s u m m e r a n d p r o v i d e d a n excellent o p p o r tunity to e x p l o r e the r e l a t i o n s h i p o f certain physical, chemical a n d biological factors to changes in the b a c t e r i a l c o m m u n i t y , to d e t e r m i n e w h a t bacterial g r o u p s s h o u l d be m e a s u r e d a n d to decide on m e t h o d s o f isolation. TABLE 1. PHYSICAL DATA*
Length (l) Depth (max at dam) Area (A) Circumference (C) Volumet Mean breadth (Aft) Shore development c/(2V'rtA) Population served
1,524 m 2.6 m 12.9 Acres 11,188 ft (3410 m) 2,000,000 ft 3 (15,000,000 gal) 112.7 ft (342.5 m) 4.2 20-30,000
* As determined from aerial photos and direct measurement. t Cubic feet and gallons rounded to nearest million. TABLE2. LIGHT, TEMPERATURE, FLOW AND BACTERIALDATA 1966-67 Range Parameter Langleys* (per day) Air T °C Max Min Av.l" Retention time + (hr) Flow (ft3/sec) Water T °C Bacteria§ (count or MPN/ml) Coliforms EC mFC KF mE 35G 20C
Pond or inlet 24-144 --1.1-36.1 --15.6-17.8 --8.3-23.6 48.2-13.1 9.7-22.7 9.5-22.0 4.3 T-240T 2.3T-240T 17-0.64T 660-13.3T 600-6.24T 2.18T-3.72M 8.05T-13.9M
Average Outlet
11.5-42.5 8.5-25.0 24-46T 43-9.3T 0.6-2.2T 0.1-1.23T 0.9-1.29T 17.5T-4.35M 1.81T-12.8M
Pond or inlet
Outlet
90.9 22.5 5.2 13.9 20.0 16.1 16.8
27.2 16.7
31T 13T 1.2T
2.6T 2T 760T 2.3M
1.4T 1.IT 54 26 77 350T 1.4M
* Theoretical value for the day of sample as determined from weather station data for this area. t This value is the average of Max-Min temperatures for the day of sample. ~. Pond volume ft3/outlet fta/sec × 3600. § Average counts per ml determined from the logarithmic average.
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
343
This report is the first in a series on the bacterial ecology of sewage stabilization ponds and deals primarily with a 2-yr study of the inter-relationship of certain physical factors and the survival of several groups of bacteria and a 1-yr follow-up study o f the "clean-up" of the pond after sewage was diverted to the newly completed permanent treatment ponds. MATERIALS AND METHODS Physical measurements
Water temperature at the time of sampling was determined by a mercury stem immersion thermometer at a depth of about 15 cm. Air temperature minima and maxima were obtained from the official weather reports for a station 1.6 km East and 3.2 km North at about the same elevation. Temperature averages were calculated from this data. Langleys per day, a measure of light intensity, were integrated from a plot of Langleys per minute per day of the year for this latitude. These values do not take into account cloud cover effects. Pond Inlet flow rates were obtained from continuous strip chart recording devices at the sewer outfalls. Outlet flow rates were determined from continuous recording devices and direct measurement by plotting on calibration curves (HYATT et al., 1965) for the outlet structures. Pond size and volume determinations were made from a combination of aerial photos and on-site measurements. Sampling
Inlet and Outlet planktonic samples were collected twice weekly within 15 min of each other in sterile containers and returned immediately to the laboratory. Analysis was begun within 30 min of sampling and no special precautions were taken in holding the samples. Samples were collected from 15 to 30 cm below the water surface moving up and across the stream so that a representative sample of the stream flow would be taken. Turbulence at the inlet due to stream fall and configuration resulted in complete mixing and 100 ml was considered a representative sample. At the outlet 500 ml taken in the same manner was considered a representative sample since the opening was about 0.8 m deep and 1.5 m wide. Because of the flow rate and sample time the sample representated on the average about 80 cubic feet of water at the inlet and outlet. Bacterial measurements
All media unless otherwise stated were Difco products. Coliforms were determined in lactose broth by 3 tube M P N method with confirmation in brilliant green lactose bile broth in accordance with Standard Methods (APHA, 1965). Fecal Escherichia coli (EC) was determined by subculture of positive presumptive coliform tubes into EC broth with incubation in water bath at 44.5°-¢-0.5°C for 24 hr (APHA, 1965). The membrane filter mFC medium and method (mFC) of GELDREICH et aL (1965) was also used as a direct assay o f this group. Enterococci at the outlet were determined by the membrane filter method with incubation on the K F agar medium (KF) of KENNER et aL (1961) and mEnterococcus agar (APHA, 1965) plates (mE). At the Inlet a spot dilution method directly on the surface of these media was used (Post et aL, 1963). Total counts were made by the pour plate technique (APHA, 1965) using plate count agar. Incubation of duplicate plates of several dilutions was made at 35°C and 20°C. The 35°C plates were incubated for 48 hr (APHA, 1965) while the
344
F.J. POST
20°C plates were incubated for 1 week. This latter incubation time was found to increase the total count many times over the recommended method (APHA, 1965). All bacterial counts and M P N values were recorded as count per ml, converted to logarithms and coded on IBM cards. Multiple regression analyses and other statistical determinations were made using the IBM 360 computer and programs developed by the staff of the Utah State University Computer Center. Occasional microscopic observations in phase contrast were made on samples of the biological mat floating on the surface of the pond. RESULTS AND DISCUSSION The pond lies at an elevation of 1372 m (4500 ft) on the floor of a large valley that was once an arm of Lake Bonneville Of which the Great Salt Lake is the last remnant. Physical parameters of size, shape and volume will be found in TABLE 1. The bottom was impervious clay, smooth and regular in cross-section tapering gently toward the centre of the channel. For the first two-thirds of its length (FIG. 1) it was very narrow and shallow, reaching a width of 10 m and a depth of about 1.5 m. From there to the dam the channel widened considerably, the slope to the center flattened and the channel tapered gently to its deepest point 2.6 m at the dam. It was supplied with water from the sewers of Logan, from numerous springs upwelling around the pond periphery (FIG. 1) and from wells serving the Utah State Fish Hatchery. The flow from the Logan sewers (about 11 fta/sec in winter) was nearly doubled during the summer irrigation season by infiltration from numerous canals flowing through the city. The springs and the hatchery during the summer doubled the sewer flow volume resulting in larger effluent than influent flow rates, TABLE 2. During the winter, spring flow slows and hatchery use is markedly lower but these were still larger flow rates than the city sewage at the same time. Occasionally irrigation diversion of the sewage stream above the inlet occurred greatly reducing the flow. This diverted water by-passed the pond entirely. These variations in sources produced widely fluctuating flow rates at the outlet and consequently highly variable retention times. The inlet flow rates were relatively stable except when infrequently diverted. During the mid-October to mid-May period the pond was normally empty and rain and snow melt added to the flow. The pond was full for only a short period during the winter, emptied when it was found that the warm waters created a serious fog problem along the adjacent highway. During the summer rainfall is negligible, with rare exceptions, and did not contribute materially to the flow. The temperature of the water flowing into the ponds was quite stable. The sewage from the city was not lower than 9.5°C TABLE2, when it entered the pond even in the coldest part of the year (Jam-Feb.). The spring and well water is a constant 10-12°C all year although flow is reduced in the winter. When the pond was full during the winter it did not freeze, in fact did not go below 8.5°C even though the coldest sample day was --15.6°C and there were colder days during non-sampling periods. When the pond was first filled during mid-May a biological mat gradually developed over a large part of the surface. Microscopic examination indicated the mat was predominantly blue-green algae of the Oscillatoria type accompanied by nonpigmented filaments of the Beggiatoa type with sulfur inclusions. These were a prominent part of the mat for most of the summer. Diatoms also occurred changing species
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
345
with the season. Planktonic algae did occur but were not followed up. Algae attached to the sides and structures were predominantly Oscillatoria types. In early spring and again in late fall when the water temperature was between 10-15°C, a cottony bloom of Sphaerotilus natans appeared on all submerged structures at the outlet. This organism was always present in the inlet in about the same biomass but was usually not in evidence at the outlet. Whether this was a temperature-competition directed or a nutrient directed bloom was not determined. When the logarithms of the bacterial counts were plotted against time of year, there was an obvious cyclic pattern. At the inlet, counts appeared to fluctuate very little, being higher in the winter and lower in the summer in the 1966 period but quite constant during 1967. Only the K F enterococcus and fecal E. coli (EC) counts are presented here, FIGS. 2 and 3; however, the observations applied to all the bacterial groups measured.
June I
Aug. I
Oct. I
1966
Dec. I
IMtor, I
Date
May 1
July I
Sept, I
Nov I
1967
FIG. 2. Outlet temperature and KF-enterococcus counts for 1966-1967. Curves were fitted by eye.
At the outlet the summer-winter difference was quite extreme for some groups but not so noticeable for others. The 20°C plate count showed the least summer decline, much less than one log cycle, and during the winter, no decline at all. The 35°C plate count showed only slightly more summer decline than the 20°C count but was otherwise the same. Data from the curves of best fit, as in FIGS. 2 and 3 were used in calculating percentage reduction of the groups during the peaks of the summer and the winter, TABLE 3. As reported by many investigators, the enterococci, as measured by either the K F or mE techniques, exhibited the greatest percentage reduction in both years. The fecal E. coli as measured by either the EC method or the mFC method showed high percentages of reduction. The mFC method presented some rather serious WATER 4/5--B
346
F . J . POST 0.5
50
05
4
4 , 0 - - - -
W o
O5
q
30
0.~
2.0 -
-
-
0.5 dune
4
4 Aug I
Dec. I4
Oci. I
Mar. l
May
July I
Date
1966
Sepl. I
Nov. I
1967
FiG. 3. Outlet temperatureand EC-fecalcoliform MPN per ml for 1966-1967. Curves were fitted by eye
TABLE 3. PERCENTAGE REDUCTION OF VARIOUS BACTERIAL GROUPS*
Coliforms
1966 Max Min 1967 Max Min
~ reduction ~o reduction Y/ooreduction ~ reduction
87.2 50.0 85.0 63.7
Fecal E. coli EC mFC
99.4 71.9 97.5 72.0
99.9 80.0 98.3 82.1
Enterococci mE KF
99.1 71.4 99.2 77.1
Total count 35°C 20°C
99.99 87.50 99.92 90.00
80.3 0.0 71.4 0.0
* Reductions based on curves of best fit for data plotted as in FIGS. 2 and 3.
TABLE 4. PERCENTAGE RATIOS OF SELECTED GROUPS OF BACTERIA IN INLET AND OUTLET WATERS AS DETERMINED FROM MEAN COUNTS OF THE GROUPS
First group as a percentage of second group Groups compared
EC-coliforms KF-coliforms KF-EC mE-EC coliforms-20° 350-20 ° mFC-EC
Inlet
Outlet
Per cent change
45.0 8.7 20.9 15.9 1.4 36.4 14.2
26,8 0,6 2,0 6.5 0.3 26.6 4.9
--40.5 --93.1 --90.5 --59.1 --78.6 --27.7 --66.0
71.3 0.0 75.4 0.0
E c o l o g y of Selected Bacteria in a Small I n t e r m i t t e n t Sewage P o n d
347
technical difficulties with counts consistently about 10 per cent or less of the EC method, TABLE4, and almost always outside the 95 per cent confidence limits of the E C - M P N number. However, the counts seemed more or less internally consistent and apparently measured a specific group or strain of fecal E. coli. The mFC medium frequently failed to grow E. coli at dilutions where they were known to be present. Attempts to overcome these problems by using different lots of medium from two different sources (Difco and Millipore) and various modifications of incubation (thermostatically controlled water baths, water jacketed incubators) produced the same results regardless of medium source or incubation conditions. The method was finally abandoned because of these difficulties. Results of the mFC medium are based on 69 samples and are not included in some of the analyses. The general coliform group showed much less reduction, intermediate between the special fecal groups and the total counts during the summer but showing considerable reduction in the winter. Based on the data in TABLE 3 and FIG. 2 and 3, it is felt that the winter values may represent loss due to settling within the pond although this point is not clear. Reduction due to settling should at least not exceed this maximum value. All the physical parameters were tested against the various counts by regression analysis postulating linear models. At the inlet, no model tested indicated very strong relationships in spite of changing flow rates, temperature, etc. No transformation of the data (logs, square roots, reciprocals, etc.) improved correlation. At the outlet, however, the single factor showing greatest correlation and the model accounting for the most variation in count was the temperature of the outlet water. Of the various models studied the cube of the temperature was significant at the 5 per cent level by F test and gave a high R 2 and correlation coefficient (R) for five of the groups. With the addition of the fourth power of the temperature the other two counts were also significant, FIG. 4. The simple linear model of count vs. temperature was also significant, at the 5 per cent level but the r 2 and r values were lower. The only count which did not conform was the 35°C plate count. With this count all parameters tested yielded very low R 2 values and correlation coefficients less than 0.50. Apparently this group behaved differently than the others for some obscure reason. The cubic equation, TABLE5, for the K F and EC counts have been plotted in FIGS. 4 and 5. The simple linear model and cubic model lines coincide between 15°C and 20°C. Based on more recent work in the new ponds the extrapolation of the cubic equation to lower temperatures seems more reasonable than that of the simple linear TABLE 5. MODELS FOR TEMPERATURE VS. COUNT AS DETERMINED BY MULTIPLE REGRESSION ANALYSIS
M o d e l (log~o) lo g log log log log log log
KF = coliforms= EC = 35 ° = mFC = mE = 20 ° =
3.01-0.000302T 3 4.17-0.000101T a 3.734).000126T 3 5.76-0.00004T 3 2.78-0.000158T 3 3.294).000506T 3 + 0.000013T ~ 6.75-0.000269 a + 0.000008T*
R2 0.733 0.369 0.566 0.181 0.417 0.717 0.506
Correlation coefficient --0.857 --0.608 --0.752 --0.426 --0.646 --0.847 --0.711
348
F.J. POST
3.0 0.5--
• ....,.~e~-o "~~ii'~e ~ e D~
2.0 "
• 1\.'
I.C
•
"o
0.~
~eee
0.5
0
5
IO
15
Temperature, *C
20
2:,5
FIo. 4. KF-enterococcus count and outlet water temperature. Equation for curve of best fit determined from multiple regression analysis using the quartic model. The equation logto KF = 3.01 --0.000302T3 used to plot the curve is significant at the 5 per cent level by F-test with a correlation coefficient of --0.857 and a regression coefficient (R2) of 0.733. model. Except for the 35°C count, the models in TABLE 5 would be considered good predictors by the predictor adequacy theory of Wetz (6) since the F values for the models exceed the tabular values by more than four times. A multiple regression analysis o f all o f the physical parameters against outlet water temperature produced the not too unexpected relationship expressed in Flo. 6. The equation: Outlet T------6.0+0.95 Inlet T + 0 . 0 4 Langleys +0.14 Air T Max gave an R 2 of 0.931 and a correlation coefficient of 0.964. The addition o f other variables was not significant at the 5 per cent level by partial F-test. The wide fluctuation in flow rate o f the outlet and consequent variation in the retention time provided an opportunity to test the formulation of MALINA and YOUSEF (1964) for coliform die-away N/No % = IO0/KR+ 1 where N = n u m b e r per ml at inlet, N o = n u m b e r per ml at outlet, R = r e t e n t i o n time, and K--velocity constant. Linear transformation of the equation indicated an R 2 value of 0.2 or less for all the groups measured suggesting that this equation does not apply or that the variability in retention time was too small to give a meaningful relationship. This point needs further study. The relative proportion of the various groups changes during residence in the pond, TABLE 4. The EC-coliform ratios are the only comparable ones methodologically
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
349
4.0
n,mee
•
i"
i
I •
• •
0,5
.
e•e•
I ,
,,- .
i¸.
30
•
"''7 '
Q
•
N
.
:.
J. •
O tad
."x'""
~e
•
•
•
0.5
2.0
0.5
I0
5
I0 Tempera'lure,
15
20
25
('C
FiG. 5. EC-fecal coliform and outlet water temperature. Equation for curve of best fit determined from multiple regression analysis using the quartic model The equation log,o EC = 3.73 --0.000126T 3 used to plot the curve is significant at the 5 per cent level by F-test with a correlation coefficient of --0.752 and a regression coefficient (R 2) of 0.566. to the Lebanon study (HORNIG et aL, 1964). The raw sewage ratio is slightly higher than reported in that study but the pond outlet ratios are comparable. All the comparisons presented here indicate a more rapid die-away of the special fecal indicator groups even though the retention time of the pond is only 24 hr or so. The group showing the greatest die-away during this period was the fecal streptococci as measured by the K F method. Regression analysis indicates some differences in ratios depending on time o f year. This data will be further analysed and presented in a later paper. After sewage was diverted to the new ponds samples were taken for analysis once a month from the stream or pond in order to follow "clean-up" since the impoundment had been receiving settled sewage for a number of years. The number of samples was too small for extensive analysis but, TABLE 6, all bacterial groups immediately dropped to fairly low levels and during the period when the pond was full, the fecal groups (EC, K F and mE) virtually disappeared. A complication arose in this part of the study when it was found that the Fish Hatchery, FIG. l, discharged raw sewage into the pond representing four permanent residents and 6-8 daytime employees. The figures in TABLE 6 may represent this pollution source as well as release f r o m pond bottom. The counts of all groups were quite consistent during the period the pond was full. The highest counts were those at 20°C incubation probably reflecting the entrance o f organic matter to the pond from fish rearing operations at the hatchery. The results o f this preliminary study suggest a high degree of relationship between
350
F.J. POST TABLE 6. BACTERIAL COUNTS IN THE POND DURING THE "CLEAN U P " PERIOD WHEN POND WAS FULL
Parameter
Range
Average
Coliforms EC KF* mE* 35 count 20 count
4.3-1 I0 0°2.3 001 0-2.4 28005.2T 50T-3.31T
21 0.3 0.13 0.009 1650 1.33T
* A zero count indicates no bacteria found in 100 ml of sample.
,
\ +0.954
+o~6r
\
I
It[~
I
[~1+o9~4 /
[_~..~/- -o.~ -0646"
I
~
-o.4~
+0 914
- 0~'847.~'~
FIG. 6. Multiple regression correlation coefficients between dominant physical factors and outlet water temperature and outlet water temperature and bacterial counts. the disappearance of certain bacterial groups in this oxidation pond and the temperature of the water. This apparent correlation, however, must be treated with caution since a high correlation coefficient does not necessarily imply a causal relationship between the two. A number of samples of raw inlet sewage were collected during the course of this study and stored at 20°C and 25°C, in the presence and absence of light for the m a x i m u m retention time of the pond with little or no change in the counts o f the various groups. This suggests that temperature itself is not the cause of the reduction but is very likely related to what produces the decline. A possibility is the biological activity stimulated by air temperature and light energy with the water temperature as simply a reflection of these effects. The specific cause of the reduction is not known. GAMESON and SAXON (1967) have suggested that daylight causes a direct reduction of coliforms in sea water. The data presented here suggest that this point should be further investigated in non-sea water as well. More recent work on the new ponds (unpublished) also suggests bacterial antagonism as well as algal activity.
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
351
O t h e r possibilities include b a c t e r i o p h a g e , p r o t o z o a a n d i n v e r t e b r a t e foragers. T h e g r o u p s o f special fecal i n d i c a t o r bacteria a p p a r e n t l y are m u c h m o r e sensitive to these e n v i r o n m e n t a l stresses t h a n the coliforms o r the so-called " b o d y t e m p e r a t u r e " b a c t e r i a (35°C t o t a l count). T h e t o t a l c o u n t at 20°C a p p e a r s to be a very insensitive i n d i c a t o r o f p o n d activity reflecting only the level o f n u m b e r s o f d e c o m p o s i n g b a c t e r i a in the p o n d . I t was n o t e d t h a t the species c o m p o s i t i o n represented on these plates c h a n g e d d u r i n g tenure in p o n d a n d this p o i n t is n o w being followed up in the new ponds. C h e m i c a l analyses w o u l d have given further i n f o r m a t i o n but were n o t begun until late in the s t u d y o f these ponds. The a m o u n t o f d a t a was thus limited. This r a t h e r small o x i d a t i o n p o n d with a retention time o f a b o u t 24 hr p r o d u c e d significant changes in the quality o f r a w sewage when biological activity a n d w a t e r t e m p e r a t u r e was high. I f the n a t u r e o f the causes o f these changes can be d e t e r m i n e d a n d p o s s i b l y c o n t r o l l e d it m a y be o f c o n s i d e r a b l e benefit in the design o f o x i d a t i o n p o n d s as well as u n d e r s t a n d i n g the role o f bacteria in the ecology o f the t r a n s f o r m a t i o n o f o r g a n i c matter. Acknowledgements--This study was supported by the Officer of Water Resources Research, U.S. Dept. Of Interior, Utah allocation CWRR-13.
REFERENCES A.P.H.A. (1965) Standard Methods for the Examination of Water. American Public Health Association, New York. BARNEKOW Jr., DAVIES R. G. and DAvis E. L. (1967)Biochemical Capabilities of Surface Filming, Benthic Bacteria in Fresh Water Systems. Water Resources Research Center, University of Missouri Report No. 2. COOPER R. C. (1967) Lagoon treatment of industrial wastes in Advances Toward Understanding Lagoon Behavior, Proceedings of the Third Annual Sanitary Engineering Conference, November 1966, College of Engineering, University of Missouri Extension Series No. 6. DRAPER N. R. and SMITHH. (1966) Applied Regression Analysis. Wiley, New York. GAMESONA. L. H and SAXONJ. R. (1967) Field Studies on effect of daylight or mortality of coliform bacteria. Water Research 1, 279-295. GELD~ICH E. E., CLARKH. F., HUFF C. B. and BESTL. C. (1965) Fecal coliform-organism medium for the membrane filter technique. J. Am. Wat. Wks Ass. 57~ 208-214. HALVORSONH., ISHAQUEM. and LEES, H. (1968) Microbiology of domestic wastes. I. Physiological activity of bacteria indigenous to Lagoon operations as a function of seasonal change. Can. J. MicrobioL 14, 369-376. HORNIO H. W. B., PORGES R., CLARK H. F. and COOKE W. B. Waste Stabilization Pond Study Lebanon, Ohio. Public Health Service Publication No. 999-WP-16. HYATr M. L., JOHNSON J. R. and ENGLANDJ. D. (1965)Flow Rate Measurement of Logan Outfall Effluents. Utah Water Research Laboratory, College of Engineering, Utah State University, Logan, Utah. KENNER B. Z., CLARKH. F. and KABLERP. W. (1961) Fecal Streptococci. I. Cultivation and enumeration of streptococci in surface waters. Appl. MicrobioL 9, 15-20. MALINAJr. J. F. and YOUSEFY. A. (1964) The fate of coliform organisms in waste stabilization ponds. J. War. Pollut. Control Fed. 36, 1432-1442. POSTF. J., KRISHNAMURTYG. B. and FLANAGANM. D. (1963) Influence of sodium hexamataphosphate on selected bacteria. Appl. MicrobioL 11,430-435. PUBLIC HEALTH SERVICE(1961) Waste Stabilization Lagoons. Proceedings of a Symposium, Kansas City, 1960. Public Health Service Publication No. 872.
ECOLOGY OF SELECTED BACTERIA IN A SMALL INTERMITTENT SEWAGE POND F. J. POST Department of Bacteriology and Public Health, Utah State University, Logan, Utah 84321, U.S.A.
(Received 10 November 1969) Abstraet--A small 24 hr retention time sewage oxidation pond in Logan, Utah has been studied to determine if any relationship exists between the population levels of certain bacteria and selected physical factors of the environment. A high degree of relationship has been found between outlet water temperature and certain groups of special fecal indicators. Fecal enterococci (as measured by the K F method) proved to be the most sensitive to environmental conditions with a correlation coefficient to temperature cubed of --0.857. Fecal Escherichia coli was also highly sensitive with a correlation coefficient of --0.752. This last group proved to be more sensitive than the coliform group as a whole (-0.608). INTRODUCTION
SEWAGEstabilization ponds have become a popular method for the disposal of municipal and industrial wastes in recent years. Concern over bacteria and viruses of public health importance has lead to numerous studies on the disappearance or survival of indicator bacteria and pathogens in these ponds (MALINA and YOUSEF 1964; PUBLIC HEALTH SERVICE, 1960). Few studies have been undertaken relating these changes to the overall bacterial ecology of the ponds. Factors that are related to the disappearance, growth or succession of the bacteria which directly affect the operation of the ponds and the bacteria themselves have been little studied. Reports of the last few years indicate an increasing interest in this direction, however (BARNEKOW and DAVIS, 1967; COOPER,1967; HALVORSONet al., 1968; HORNIGet al., 1964).
N /
p
t
IrricJation
I
~s~ . . . . . °.
/
[ Outlet
t,t-'-X,
(winter) ~
I
To
"-'
/o
hatchery ,,,
w
Loqan City
-
Loqan
S..~ter
River ,2 Marshy Sprino o Well
IrrkJation
FIG. 1. Map of sewage-irrigation pond, Logan, Utah (not to scale). 341
342
F . J . POST
In 1965 L o g a n , U t a h a n n o u n c e d the i n t e n t i o n o f c o n s t r u c t i n g a series o f waste s t a b i l i z a t i o n p o n d s to serve a c o m m u n i t y o f 25,000 to 30,000 people. This p r o v i d e d a n excellent o p p o r t u n i t y to s t u d y certain aspects o f the b a c t e r i a l ecology o f these p o n d s b e g i n n i n g with their inception. P r i o r to the c o m p l e t i o n o f these p o n d s ( N o v e m b e r 1967) L o g a n h a d been d i s c h a r g i n g sewage into two surface streams (FIG. 1) at the city limits w h i c h later flowed t o g e t h e r a n d thence into the L o g a n River, a t r i b u t a r y o f the Bear River flowing into the G r e a t Salt Lake. D u r i n g the s u m m e r m o n t h s ( m i d - M a y to m i d - O c t ) the sewage s t r e a m was d a m m e d i m p o u n d i n g a b o u t 15 million gallons o f w a t e r for a p p r o x i m a t e l y 20-24 h r (TABLES 1, 2). This p o n d served as a sewage s t a b i l i z a t i o n p o n d d u r i n g the s u m m e r a n d p r o v i d e d a n excellent o p p o r tunity to e x p l o r e the r e l a t i o n s h i p o f certain physical, chemical a n d biological factors to changes in the b a c t e r i a l c o m m u n i t y , to d e t e r m i n e w h a t bacterial g r o u p s s h o u l d be m e a s u r e d a n d to decide on m e t h o d s o f isolation. TABLE 1. PHYSICAL DATA*
Length (l) Depth (max at dam) Area (A) Circumference (C) Volumet Mean breadth (Aft) Shore development c/(2V'rtA) Population served
1,524 m 2.6 m 12.9 Acres 11,188 ft (3410 m) 2,000,000 ft 3 (15,000,000 gal) 112.7 ft (342.5 m) 4.2 20-30,000
* As determined from aerial photos and direct measurement. t Cubic feet and gallons rounded to nearest million. TABLE2. LIGHT, TEMPERATURE, FLOW AND BACTERIALDATA 1966-67 Range Parameter Langleys* (per day) Air T °C Max Min Av.l" Retention time + (hr) Flow (ft3/sec) Water T °C Bacteria§ (count or MPN/ml) Coliforms EC mFC KF mE 35G 20C
Pond or inlet 24-144 --1.1-36.1 --15.6-17.8 --8.3-23.6 48.2-13.1 9.7-22.7 9.5-22.0 4.3 T-240T 2.3T-240T 17-0.64T 660-13.3T 600-6.24T 2.18T-3.72M 8.05T-13.9M
Average Outlet
11.5-42.5 8.5-25.0 24-46T 43-9.3T 0.6-2.2T 0.1-1.23T 0.9-1.29T 17.5T-4.35M 1.81T-12.8M
Pond or inlet
Outlet
90.9 22.5 5.2 13.9 20.0 16.1 16.8
27.2 16.7
31T 13T 1.2T
2.6T 2T 760T 2.3M
1.4T 1.IT 54 26 77 350T 1.4M
* Theoretical value for the day of sample as determined from weather station data for this area. t This value is the average of Max-Min temperatures for the day of sample. ~. Pond volume ft3/outlet fta/sec × 3600. § Average counts per ml determined from the logarithmic average.
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
343
This report is the first in a series on the bacterial ecology of sewage stabilization ponds and deals primarily with a 2-yr study of the inter-relationship of certain physical factors and the survival of several groups of bacteria and a 1-yr follow-up study o f the "clean-up" of the pond after sewage was diverted to the newly completed permanent treatment ponds. MATERIALS AND METHODS Physical measurements
Water temperature at the time of sampling was determined by a mercury stem immersion thermometer at a depth of about 15 cm. Air temperature minima and maxima were obtained from the official weather reports for a station 1.6 km East and 3.2 km North at about the same elevation. Temperature averages were calculated from this data. Langleys per day, a measure of light intensity, were integrated from a plot of Langleys per minute per day of the year for this latitude. These values do not take into account cloud cover effects. Pond Inlet flow rates were obtained from continuous strip chart recording devices at the sewer outfalls. Outlet flow rates were determined from continuous recording devices and direct measurement by plotting on calibration curves (HYATT et al., 1965) for the outlet structures. Pond size and volume determinations were made from a combination of aerial photos and on-site measurements. Sampling
Inlet and Outlet planktonic samples were collected twice weekly within 15 min of each other in sterile containers and returned immediately to the laboratory. Analysis was begun within 30 min of sampling and no special precautions were taken in holding the samples. Samples were collected from 15 to 30 cm below the water surface moving up and across the stream so that a representative sample of the stream flow would be taken. Turbulence at the inlet due to stream fall and configuration resulted in complete mixing and 100 ml was considered a representative sample. At the outlet 500 ml taken in the same manner was considered a representative sample since the opening was about 0.8 m deep and 1.5 m wide. Because of the flow rate and sample time the sample representated on the average about 80 cubic feet of water at the inlet and outlet. Bacterial measurements
All media unless otherwise stated were Difco products. Coliforms were determined in lactose broth by 3 tube M P N method with confirmation in brilliant green lactose bile broth in accordance with Standard Methods (APHA, 1965). Fecal Escherichia coli (EC) was determined by subculture of positive presumptive coliform tubes into EC broth with incubation in water bath at 44.5°-¢-0.5°C for 24 hr (APHA, 1965). The membrane filter mFC medium and method (mFC) of GELDREICH et aL (1965) was also used as a direct assay o f this group. Enterococci at the outlet were determined by the membrane filter method with incubation on the K F agar medium (KF) of KENNER et aL (1961) and mEnterococcus agar (APHA, 1965) plates (mE). At the Inlet a spot dilution method directly on the surface of these media was used (Post et aL, 1963). Total counts were made by the pour plate technique (APHA, 1965) using plate count agar. Incubation of duplicate plates of several dilutions was made at 35°C and 20°C. The 35°C plates were incubated for 48 hr (APHA, 1965) while the
344
F.J. POST
20°C plates were incubated for 1 week. This latter incubation time was found to increase the total count many times over the recommended method (APHA, 1965). All bacterial counts and M P N values were recorded as count per ml, converted to logarithms and coded on IBM cards. Multiple regression analyses and other statistical determinations were made using the IBM 360 computer and programs developed by the staff of the Utah State University Computer Center. Occasional microscopic observations in phase contrast were made on samples of the biological mat floating on the surface of the pond. RESULTS AND DISCUSSION The pond lies at an elevation of 1372 m (4500 ft) on the floor of a large valley that was once an arm of Lake Bonneville Of which the Great Salt Lake is the last remnant. Physical parameters of size, shape and volume will be found in TABLE 1. The bottom was impervious clay, smooth and regular in cross-section tapering gently toward the centre of the channel. For the first two-thirds of its length (FIG. 1) it was very narrow and shallow, reaching a width of 10 m and a depth of about 1.5 m. From there to the dam the channel widened considerably, the slope to the center flattened and the channel tapered gently to its deepest point 2.6 m at the dam. It was supplied with water from the sewers of Logan, from numerous springs upwelling around the pond periphery (FIG. 1) and from wells serving the Utah State Fish Hatchery. The flow from the Logan sewers (about 11 fta/sec in winter) was nearly doubled during the summer irrigation season by infiltration from numerous canals flowing through the city. The springs and the hatchery during the summer doubled the sewer flow volume resulting in larger effluent than influent flow rates, TABLE 2. During the winter, spring flow slows and hatchery use is markedly lower but these were still larger flow rates than the city sewage at the same time. Occasionally irrigation diversion of the sewage stream above the inlet occurred greatly reducing the flow. This diverted water by-passed the pond entirely. These variations in sources produced widely fluctuating flow rates at the outlet and consequently highly variable retention times. The inlet flow rates were relatively stable except when infrequently diverted. During the mid-October to mid-May period the pond was normally empty and rain and snow melt added to the flow. The pond was full for only a short period during the winter, emptied when it was found that the warm waters created a serious fog problem along the adjacent highway. During the summer rainfall is negligible, with rare exceptions, and did not contribute materially to the flow. The temperature of the water flowing into the ponds was quite stable. The sewage from the city was not lower than 9.5°C TABLE2, when it entered the pond even in the coldest part of the year (Jam-Feb.). The spring and well water is a constant 10-12°C all year although flow is reduced in the winter. When the pond was full during the winter it did not freeze, in fact did not go below 8.5°C even though the coldest sample day was --15.6°C and there were colder days during non-sampling periods. When the pond was first filled during mid-May a biological mat gradually developed over a large part of the surface. Microscopic examination indicated the mat was predominantly blue-green algae of the Oscillatoria type accompanied by nonpigmented filaments of the Beggiatoa type with sulfur inclusions. These were a prominent part of the mat for most of the summer. Diatoms also occurred changing species
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
345
with the season. Planktonic algae did occur but were not followed up. Algae attached to the sides and structures were predominantly Oscillatoria types. In early spring and again in late fall when the water temperature was between 10-15°C, a cottony bloom of Sphaerotilus natans appeared on all submerged structures at the outlet. This organism was always present in the inlet in about the same biomass but was usually not in evidence at the outlet. Whether this was a temperature-competition directed or a nutrient directed bloom was not determined. When the logarithms of the bacterial counts were plotted against time of year, there was an obvious cyclic pattern. At the inlet, counts appeared to fluctuate very little, being higher in the winter and lower in the summer in the 1966 period but quite constant during 1967. Only the K F enterococcus and fecal E. coli (EC) counts are presented here, FIGS. 2 and 3; however, the observations applied to all the bacterial groups measured.
June I
Aug. I
Oct. I
1966
Dec. I
IMtor, I
Date
May 1
July I
Sept, I
Nov I
1967
FIG. 2. Outlet temperature and KF-enterococcus counts for 1966-1967. Curves were fitted by eye.
At the outlet the summer-winter difference was quite extreme for some groups but not so noticeable for others. The 20°C plate count showed the least summer decline, much less than one log cycle, and during the winter, no decline at all. The 35°C plate count showed only slightly more summer decline than the 20°C count but was otherwise the same. Data from the curves of best fit, as in FIGS. 2 and 3 were used in calculating percentage reduction of the groups during the peaks of the summer and the winter, TABLE 3. As reported by many investigators, the enterococci, as measured by either the K F or mE techniques, exhibited the greatest percentage reduction in both years. The fecal E. coli as measured by either the EC method or the mFC method showed high percentages of reduction. The mFC method presented some rather serious WATER 4/5--B
346
F . J . POST 0.5
50
05
4
4 , 0 - - - -
W o
O5
q
30
0.~
2.0 -
-
-
0.5 dune
4
4 Aug I
Dec. I4
Oci. I
Mar. l
May
July I
Date
1966
Sepl. I
Nov. I
1967
FiG. 3. Outlet temperatureand EC-fecalcoliform MPN per ml for 1966-1967. Curves were fitted by eye
TABLE 3. PERCENTAGE REDUCTION OF VARIOUS BACTERIAL GROUPS*
Coliforms
1966 Max Min 1967 Max Min
~ reduction ~o reduction Y/ooreduction ~ reduction
87.2 50.0 85.0 63.7
Fecal E. coli EC mFC
99.4 71.9 97.5 72.0
99.9 80.0 98.3 82.1
Enterococci mE KF
99.1 71.4 99.2 77.1
Total count 35°C 20°C
99.99 87.50 99.92 90.00
80.3 0.0 71.4 0.0
* Reductions based on curves of best fit for data plotted as in FIGS. 2 and 3.
TABLE 4. PERCENTAGE RATIOS OF SELECTED GROUPS OF BACTERIA IN INLET AND OUTLET WATERS AS DETERMINED FROM MEAN COUNTS OF THE GROUPS
First group as a percentage of second group Groups compared
EC-coliforms KF-coliforms KF-EC mE-EC coliforms-20° 350-20 ° mFC-EC
Inlet
Outlet
Per cent change
45.0 8.7 20.9 15.9 1.4 36.4 14.2
26,8 0,6 2,0 6.5 0.3 26.6 4.9
--40.5 --93.1 --90.5 --59.1 --78.6 --27.7 --66.0
71.3 0.0 75.4 0.0
E c o l o g y of Selected Bacteria in a Small I n t e r m i t t e n t Sewage P o n d
347
technical difficulties with counts consistently about 10 per cent or less of the EC method, TABLE4, and almost always outside the 95 per cent confidence limits of the E C - M P N number. However, the counts seemed more or less internally consistent and apparently measured a specific group or strain of fecal E. coli. The mFC medium frequently failed to grow E. coli at dilutions where they were known to be present. Attempts to overcome these problems by using different lots of medium from two different sources (Difco and Millipore) and various modifications of incubation (thermostatically controlled water baths, water jacketed incubators) produced the same results regardless of medium source or incubation conditions. The method was finally abandoned because of these difficulties. Results of the mFC medium are based on 69 samples and are not included in some of the analyses. The general coliform group showed much less reduction, intermediate between the special fecal groups and the total counts during the summer but showing considerable reduction in the winter. Based on the data in TABLE 3 and FIG. 2 and 3, it is felt that the winter values may represent loss due to settling within the pond although this point is not clear. Reduction due to settling should at least not exceed this maximum value. All the physical parameters were tested against the various counts by regression analysis postulating linear models. At the inlet, no model tested indicated very strong relationships in spite of changing flow rates, temperature, etc. No transformation of the data (logs, square roots, reciprocals, etc.) improved correlation. At the outlet, however, the single factor showing greatest correlation and the model accounting for the most variation in count was the temperature of the outlet water. Of the various models studied the cube of the temperature was significant at the 5 per cent level by F test and gave a high R 2 and correlation coefficient (R) for five of the groups. With the addition of the fourth power of the temperature the other two counts were also significant, FIG. 4. The simple linear model of count vs. temperature was also significant, at the 5 per cent level but the r 2 and r values were lower. The only count which did not conform was the 35°C plate count. With this count all parameters tested yielded very low R 2 values and correlation coefficients less than 0.50. Apparently this group behaved differently than the others for some obscure reason. The cubic equation, TABLE5, for the K F and EC counts have been plotted in FIGS. 4 and 5. The simple linear model and cubic model lines coincide between 15°C and 20°C. Based on more recent work in the new ponds the extrapolation of the cubic equation to lower temperatures seems more reasonable than that of the simple linear TABLE 5. MODELS FOR TEMPERATURE VS. COUNT AS DETERMINED BY MULTIPLE REGRESSION ANALYSIS
M o d e l (log~o) lo g log log log log log log
KF = coliforms= EC = 35 ° = mFC = mE = 20 ° =
3.01-0.000302T 3 4.17-0.000101T a 3.734).000126T 3 5.76-0.00004T 3 2.78-0.000158T 3 3.294).000506T 3 + 0.000013T ~ 6.75-0.000269 a + 0.000008T*
R2 0.733 0.369 0.566 0.181 0.417 0.717 0.506
Correlation coefficient --0.857 --0.608 --0.752 --0.426 --0.646 --0.847 --0.711
348
F.J. POST
3.0 0.5--
• ....,.~e~-o "~~ii'~e ~ e D~
2.0 "
• 1\.'
I.C
•
"o
0.~
~eee
0.5
0
5
IO
15
Temperature, *C
20
2:,5
FIo. 4. KF-enterococcus count and outlet water temperature. Equation for curve of best fit determined from multiple regression analysis using the quartic model. The equation logto KF = 3.01 --0.000302T3 used to plot the curve is significant at the 5 per cent level by F-test with a correlation coefficient of --0.857 and a regression coefficient (R2) of 0.733. model. Except for the 35°C count, the models in TABLE 5 would be considered good predictors by the predictor adequacy theory of Wetz (6) since the F values for the models exceed the tabular values by more than four times. A multiple regression analysis o f all o f the physical parameters against outlet water temperature produced the not too unexpected relationship expressed in Flo. 6. The equation: Outlet T------6.0+0.95 Inlet T + 0 . 0 4 Langleys +0.14 Air T Max gave an R 2 of 0.931 and a correlation coefficient of 0.964. The addition o f other variables was not significant at the 5 per cent level by partial F-test. The wide fluctuation in flow rate o f the outlet and consequent variation in the retention time provided an opportunity to test the formulation of MALINA and YOUSEF (1964) for coliform die-away N/No % = IO0/KR+ 1 where N = n u m b e r per ml at inlet, N o = n u m b e r per ml at outlet, R = r e t e n t i o n time, and K--velocity constant. Linear transformation of the equation indicated an R 2 value of 0.2 or less for all the groups measured suggesting that this equation does not apply or that the variability in retention time was too small to give a meaningful relationship. This point needs further study. The relative proportion of the various groups changes during residence in the pond, TABLE 4. The EC-coliform ratios are the only comparable ones methodologically
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
349
4.0
n,mee
•
i"
i
I •
• •
0,5
.
e•e•
I ,
,,- .
i¸.
30
•
"''7 '
Q
•
N
.
:.
J. •
O tad
."x'""
~e
•
•
•
0.5
2.0
0.5
I0
5
I0 Tempera'lure,
15
20
25
('C
FiG. 5. EC-fecal coliform and outlet water temperature. Equation for curve of best fit determined from multiple regression analysis using the quartic model The equation log,o EC = 3.73 --0.000126T 3 used to plot the curve is significant at the 5 per cent level by F-test with a correlation coefficient of --0.752 and a regression coefficient (R 2) of 0.566. to the Lebanon study (HORNIG et aL, 1964). The raw sewage ratio is slightly higher than reported in that study but the pond outlet ratios are comparable. All the comparisons presented here indicate a more rapid die-away of the special fecal indicator groups even though the retention time of the pond is only 24 hr or so. The group showing the greatest die-away during this period was the fecal streptococci as measured by the K F method. Regression analysis indicates some differences in ratios depending on time o f year. This data will be further analysed and presented in a later paper. After sewage was diverted to the new ponds samples were taken for analysis once a month from the stream or pond in order to follow "clean-up" since the impoundment had been receiving settled sewage for a number of years. The number of samples was too small for extensive analysis but, TABLE 6, all bacterial groups immediately dropped to fairly low levels and during the period when the pond was full, the fecal groups (EC, K F and mE) virtually disappeared. A complication arose in this part of the study when it was found that the Fish Hatchery, FIG. l, discharged raw sewage into the pond representing four permanent residents and 6-8 daytime employees. The figures in TABLE 6 may represent this pollution source as well as release f r o m pond bottom. The counts of all groups were quite consistent during the period the pond was full. The highest counts were those at 20°C incubation probably reflecting the entrance o f organic matter to the pond from fish rearing operations at the hatchery. The results o f this preliminary study suggest a high degree of relationship between
350
F.J. POST TABLE 6. BACTERIAL COUNTS IN THE POND DURING THE "CLEAN U P " PERIOD WHEN POND WAS FULL
Parameter
Range
Average
Coliforms EC KF* mE* 35 count 20 count
4.3-1 I0 0°2.3 001 0-2.4 28005.2T 50T-3.31T
21 0.3 0.13 0.009 1650 1.33T
* A zero count indicates no bacteria found in 100 ml of sample.
,
\ +0.954
+o~6r
\
I
It[~
I
[~1+o9~4 /
[_~..~/- -o.~ -0646"
I
~
-o.4~
+0 914
- 0~'847.~'~
FIG. 6. Multiple regression correlation coefficients between dominant physical factors and outlet water temperature and outlet water temperature and bacterial counts. the disappearance of certain bacterial groups in this oxidation pond and the temperature of the water. This apparent correlation, however, must be treated with caution since a high correlation coefficient does not necessarily imply a causal relationship between the two. A number of samples of raw inlet sewage were collected during the course of this study and stored at 20°C and 25°C, in the presence and absence of light for the m a x i m u m retention time of the pond with little or no change in the counts o f the various groups. This suggests that temperature itself is not the cause of the reduction but is very likely related to what produces the decline. A possibility is the biological activity stimulated by air temperature and light energy with the water temperature as simply a reflection of these effects. The specific cause of the reduction is not known. GAMESON and SAXON (1967) have suggested that daylight causes a direct reduction of coliforms in sea water. The data presented here suggest that this point should be further investigated in non-sea water as well. More recent work on the new ponds (unpublished) also suggests bacterial antagonism as well as algal activity.
Ecology of Selected Bacteria in a Small Intermittent Sewage Pond
351
O t h e r possibilities include b a c t e r i o p h a g e , p r o t o z o a a n d i n v e r t e b r a t e foragers. T h e g r o u p s o f special fecal i n d i c a t o r bacteria a p p a r e n t l y are m u c h m o r e sensitive to these e n v i r o n m e n t a l stresses t h a n the coliforms o r the so-called " b o d y t e m p e r a t u r e " b a c t e r i a (35°C t o t a l count). T h e t o t a l c o u n t at 20°C a p p e a r s to be a very insensitive i n d i c a t o r o f p o n d activity reflecting only the level o f n u m b e r s o f d e c o m p o s i n g b a c t e r i a in the p o n d . I t was n o t e d t h a t the species c o m p o s i t i o n represented on these plates c h a n g e d d u r i n g tenure in p o n d a n d this p o i n t is n o w being followed up in the new ponds. C h e m i c a l analyses w o u l d have given further i n f o r m a t i o n but were n o t begun until late in the s t u d y o f these ponds. The a m o u n t o f d a t a was thus limited. This r a t h e r small o x i d a t i o n p o n d with a retention time o f a b o u t 24 hr p r o d u c e d significant changes in the quality o f r a w sewage when biological activity a n d w a t e r t e m p e r a t u r e was high. I f the n a t u r e o f the causes o f these changes can be d e t e r m i n e d a n d p o s s i b l y c o n t r o l l e d it m a y be o f c o n s i d e r a b l e benefit in the design o f o x i d a t i o n p o n d s as well as u n d e r s t a n d i n g the role o f bacteria in the ecology o f the t r a n s f o r m a t i o n o f o r g a n i c matter. Acknowledgements--This study was supported by the Officer of Water Resources Research, U.S. Dept. Of Interior, Utah allocation CWRR-13.
REFERENCES A.P.H.A. (1965) Standard Methods for the Examination of Water. American Public Health Association, New York. BARNEKOW Jr., DAVIES R. G. and DAvis E. L. (1967)Biochemical Capabilities of Surface Filming, Benthic Bacteria in Fresh Water Systems. Water Resources Research Center, University of Missouri Report No. 2. COOPER R. C. (1967) Lagoon treatment of industrial wastes in Advances Toward Understanding Lagoon Behavior, Proceedings of the Third Annual Sanitary Engineering Conference, November 1966, College of Engineering, University of Missouri Extension Series No. 6. DRAPER N. R. and SMITHH. (1966) Applied Regression Analysis. Wiley, New York. GAMESONA. L. H and SAXONJ. R. (1967) Field Studies on effect of daylight or mortality of coliform bacteria. Water Research 1, 279-295. GELD~ICH E. E., CLARKH. F., HUFF C. B. and BESTL. C. (1965) Fecal coliform-organism medium for the membrane filter technique. J. Am. Wat. Wks Ass. 57~ 208-214. HALVORSONH., ISHAQUEM. and LEES, H. (1968) Microbiology of domestic wastes. I. Physiological activity of bacteria indigenous to Lagoon operations as a function of seasonal change. Can. J. MicrobioL 14, 369-376. HORNIO H. W. B., PORGES R., CLARK H. F. and COOKE W. B. Waste Stabilization Pond Study Lebanon, Ohio. Public Health Service Publication No. 999-WP-16. HYATr M. L., JOHNSON J. R. and ENGLANDJ. D. (1965)Flow Rate Measurement of Logan Outfall Effluents. Utah Water Research Laboratory, College of Engineering, Utah State University, Logan, Utah. KENNER B. Z., CLARKH. F. and KABLERP. W. (1961) Fecal Streptococci. I. Cultivation and enumeration of streptococci in surface waters. Appl. MicrobioL 9, 15-20. MALINAJr. J. F. and YOUSEFY. A. (1964) The fate of coliform organisms in waste stabilization ponds. J. War. Pollut. Control Fed. 36, 1432-1442. POSTF. J., KRISHNAMURTYG. B. and FLANAGANM. D. (1963) Influence of sodium hexamataphosphate on selected bacteria. Appl. MicrobioL 11,430-435. PUBLIC HEALTH SERVICE(1961) Waste Stabilization Lagoons. Proceedings of a Symposium, Kansas City, 1960. Public Health Service Publication No. 872.