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The relationship between viability and respiration rate in the activated sludge process
Water Research Vol. I 1. pp. 575 to 578. PergamonPre~,;1977 Printedm Great Britain
THE RELATIONSHIP BETWEEN VIABILITY A N D RESPIRATION RATE IN THE ACTIVATED SLUDGE PROCESS I. WAI,KER and M. DAVIES Department of Biology, University of York, Heslington, York YOI 5DD, England (Received 5 September 1976) Abstract- .The possibility that non-viable bacteria could be responsible for a proportion of the activity of an activated sludge, was discussed by Jones (1973). Following this, the work reported here was undertaken to observe the relationship between the respiratory activity and the unit viability of activated sludges cultured at various net growth rates. Both viable counts and respiration rates increased to maximum values at a growth rate of 0.1 days-~. However. it was found that the apparent respiration rate per viable cell was not constant, as reported by Weddle and Jenkins (1971). but that it increased at low growth rates. At a growth rate of 0.1 days-~ the observed activity was six times that expected from the observed viability. The results are explained by attributing part of the activity to non-viable bacteria.
INTRODUCTION
Work done by Postgate and Hunter (1962), using cultures of Aerobacter aerogenes grown in a chemostat with replacement times of up to 11 days, showed that only 37.8~o of the bacteria were demonstrably viable at very low growth rates. Weddle and Jenkins (1971} have shown that the numbers of viable ceils m g volatile suspended solids (unit viability) in activated sludge vanes with respect to the mean cell residence time of the system. Banks et al. (1976) found that the viability of sludges from ten different municipal activated sludge units ranged from 0.23 × 1()~ to 5.9 × 10 ~ cells g ~ of sludge (dry weight): this is approximately 2-60°/, viability. Jones (1973) suggested that the presence of nonviable but active bacteria could account for the d i s erepancy between the observed metabolic activity of a sludge and the lower value predicted from the numbers of bacteria shown to be present (Department of the Environment, 1971). The aim of the work reported here was to study the relationship between the activity and viability of sludges cultured at different growth rates, using a respirometer fitted with an oxygen electrode. The potential respiration rate (obtained when substrate is not limitingl was preferred as a measure of activity, to the rate in situ where substrate concentration is low. To allow for possible variations in floc structure each sample of sludge was exposed to a range of sonication periods before viability determinations wcre made. The sludge used was cultured from, and grown on. a domestic sewage in order to produce a naturally adapted population. MATERIALS AND METHODS
Culture system. The activated sludge unit consisted of a two tank aeration system of 3 I capacity, each tank contained a four inch stone air diffuser, and a 3 1 settlement
tank. The latter was a modified funnel fitted with an overflow weir and a rotating scraper chain to prevent sludge from accumulating on the sides (Jones, 1965). Sewage from the University's sewerage system was collected daily at 10.45am and settled for 18h before use. It was stored under refrigeration and fed continuously by peristaltic pump to the first aeration tank at a rate of 1.21 h - t ; sludge was recycled from the settlement tank at 0.8 I h *. The net growth rate of the sludge was controlled b) wasting from the aeration tank. as described by Garrett (1958). Calculation of the mean cell residence time is based on the assumption that the settlement tank contents has an average solids concentration equivalent to the mixed liquors solid's concentration (Burchett and Tchobanoglous, 1974). The volume of mixed liquor wasted each day from the aeration tank was calculated from the formula (~, + t'~ /"~ . . . . . . . 0r
F,.' .~'~
I X
(11
where: F~. = volume of mixed liquor to be removed each day, 1, X = concentration of solids (dry weightl in the mixed liquor, g. 1- ~, Va = volume of aeration system. 1. V, = volume of settlement tank. 1, F~ = tlow rate of effluent leaving the settlement tank, i.d-I X~ = concentration of solids (dry weight~ in the effluent. g.1-1. 0~ = required mean cell residence time Im.c.r.t.I, days, (equivalent to I/It where u = net growth rate, days- t). When F,. was less than 0.51 mixed liquor was wasted manually once a day; when F,,. was greater than 0.51 mixed liquor was automatically wasted by peristaltic pump once an hour. Viability determination. A five ml sample of mixed liquor was diluted ten-fold with sodium tripolyphosphate buffer (Pike et al., 1972), and placed in an ice cooled vessel. The sample was then sonicated using a Dawc Soniprobe (Model No. 1130) at a scale reading of 3.6. determined to be the optimum power output by Banks and Walker (1976). One ml samples were removed from the vessel 40, 60 and 80sec after commencement of sonication: the samples were serially diluted in polyphosphatc buffer and
575
I. WALKER and M. DAVIES
576
duplicate 0.1 ml vol of the 10 -~ and 10 " dilutions were spread on to agar plates. The medium used was C . G Y agar (casitone 5 g ' l - ' , glycerol 5 g . l '. yeast extract I g. 1- J, oxoid agar No. 3 13 g.l- ~) as recommended for maximal recovery by Pike et al. (19721. The colonies were counted after seven days incubation at 2 r C . Results are expressed as unit viability, that is the maximum viable cell count/g dry weight of mixed liquor suspended solids. Respiration rate determination. Oxygen uptake rates were measured in a Rank respirometer. 7 ml capacity, at 20C. Settled sewage was sampled from the holding tank, filtered through a "Millipore" membrane with a pore diameter of 0.45/~m and then aerated at room temperature for 10 rain, Five ml of this sewage was added to the incubation vessel and was allowed to equilibrate at 2 0 C after which 2 ml of the mixed liquor to be assayed was added: the vessel was sealed from the atmosphere and the fall in oxygen tension of the mixture was recorded. After 4-.Stain the incubation was stopped and 5 ml of the sample was withdrawn, the dry weight of this sample was determined. Four incubations were carried out for each mixed liquor assayed and the mean of these values is expressed as mg oxygen utilized g- ' of mixed liquor {dry weight) h- '. Each result reported is the mean of four determinations obtained when the activated sludge units were stable at the required growth rate. The units were classified as unstable if the C.O.D. of the sewage diverged by more than 20% from the mean of the previous three days. In all cases, excepting the lowest growth rate, the units had been operating at the maintained growth rate for at least one m.c.r.t. before measurements were recorded.
Table I. Concentration of mixed liquor suspended solids at different values of net growth rate 0,-~, days J
MLSS. mg.I :
0.0075 0.0480 0.(090 0.1520 O.1661) o. 1750 O.2OOO
7815 (~550 4() I 0 2825 245(I 28o0 232o 17(X)
0.3080 0.4090
[490 I l)l8
0.4330 1.0040
1260
2.7000
571)
It was found that both viable ceil counts a n d respiration rates increased with increasing values of 0; to reach a m a x i m u m value where 0¢- ~ = 0.7-O.~ d a y - ' (Fig. 1). Viable cell counts increased slightly at the lowest growth rates a n d then rose rapidly from 0.5 x 10 ~ cells g - ' to a m a x i m u m of 4.5 x 1('0 ~ cells g - ~. Respiration rates increased rapidly from t 3.2 mg O 2 g - ' h ' at the lowest net growth rate to a maxim u m of 70 mg 0 2 g - ~ h - ~. The relationship between net growth rate and thc a p p a r e n t respiration rate per viable cell counted, R a. is shown in Fig. 2. At the higher growth rates R, was constant at a value of 1.46 x 1 0 - ' : mg O , consumed per viable bacterium h - t . As the growth rate fell below 0.4 d a y - ' R, increased approximately six fold to a value of 9.5 × 10 -~-' mg 0 2 consumed per viable bacterium h - L where 0 [ ~ = 0.1 d a y ', and then fell toward zero,
RESULTS The m e a n C.O.D. of the sewage fed to the units on the days of experimentation was 290 nag. 1 - *, ranging from 260 to 3 1 5 m g ' l - L Effluent C.O.D, varied from 20 to 50 mg. 1-~ a n d effluent suspended solids varied from 10 to 3 0 r a g - I - ~ . Net growth rate, 0 [ ~, was calculated from a rearrangement of equation (1) a n d was 0,0075 day-~ at its lowest and 2.7 d a y at highest. The concentration of solids in the aeration system decreased with increasing values of 0,: Table 1.
D I S C U S S I O N
F r o m Fig. 1 it would appear that above a net growth rate of a r o u n d 0.9 d a y t the viable cell counts
b
60
L
¢3"
.c
°/ 50
•
,.c
(5 ~ 4 0
.
g C3 0.
30
20
J 0
4
>i
0
1 0
---
0.25
g --
I 0.50 Net
1
1
0.75 qrowth
J__l
J.O0 rate,
L25 8~',
i
,1 2.50
3.00
days-'
Fig. t. Effect of net growth rate on unit viability, O, and on respiration rate, 0.
Relationship between viability and respiration rate
577
'0 x
J~ x
6 v x c~ 0
4
O
1
I 0.25
I 0.50 Net
I
I
0.75 growth
I
I.O0
~ c I,
rote,
.
~.25
.
.
.
I
I
2.50
2 7O
c l o y s -I
Fig. 2. Effect of net growth rate on the apparent respiration rate per viable bacterium. Ra.
cannot be increased. This would indicate that the culture is I(X)~,~ viable and compares well with the data of Postgate and Hunter (1962) and Weddle and Jenkins (1971). Thc calculated dry weight of an average bacterium, at a net growth rate of 2.7 day- ~, of 2.22 x 10 ~-'g is not unreasonable if the culture is 10~)",, viable and the respiration rate of 1.46 x 1 0 - 1 2 m g O 2 bacterium -~ h t at the ~ m c net growth rate, is in good agreement with the rate obtained by Wcddlc and Jenkins (1971). 1.01 x 10 ~2 mg 02 bactcrium-~ h ~. Below a net growth of 0.9 day : the bacteria are probably unable to maintain their viability longer than the mean cell residence time. resulting in a proportion of the bacteria in the culture system being non-viable. If viable bacteria are rcsponsiblc for the total respiratory activity of a sludge then it is reasonable to expect that the observed respiration rate per viable cell should be constant over a wide rangc of net growth rates. This is a conclusion drawn by Weddle and Jenkins ( 1971 ). although their standard deviation is more than 60",, of their mean. Figure 2 contradicts this expectation, with viable bacteria appearing to respire more than six times faster at a net growth rate of 0.1 da 5- i. than at a net growth rate of 2.7 day-~. it is unlikely that this apparent increase in respiratory activity at lo~s. net growth rates is due to an increase in the maintenance requirements of the bacteria or to a "'slip" type of activity due to substrate limitations (Ncijssel and Tempest. 1976). These reactions cannot explain the fall in the apparent respiration rates at the lowest net growth rates. The most probable reason for the observations is that at the lower net growth rates a proportion of the respiratory activity can bc attributed to dying, non-viable bacteria. The process of bacterial death in slow growing continuous cultures is not clearly understood; Postgate's work (1962) has indicated that "dead" cells retain their osmotic integrity and several authors have reported that loss of viability is not necessarily associated with
loss of biochemical activity (Wooldridgc and Standfast, 1936; Stephenson, 1928). By assuming that the respiration rate of an average bacterium is 1.46 x 1 0 - ~ " m g 0 2 h ~ and that this value is independent of the net growth rate. the respiratory activity of the viable bacteria can be calculated: subtraction of this from the total activity should give an estimate of the activity due to non-viable bacteria. It can be seen in Fig. 3 that. at a growth rate of 0.1 d a y ~ over 80~o of the total respiration could bc attributed to non-viable bacteria. It is considered that the shape of Fig. 3 is duc to the death and decay rates of viable and non-viable bacteria increasing as the sludge net growth rate decreases. The total number of non-viable cells would increase but the proportion of these that arc active decreases. The changes are likely to bc due to increases in the maintenance requirement of the culture: as the solids level increases the total amount of substratc required to maintain all the cells in an active state increases, at some point the substratc supplied is insufficient to meet the needs of the culture and the rate of decay increases. .oo
o
80
o
./.
"6 6C
40
If o
0.2
0.4 Net
0.6
08
g r o w t h rate,
e
,O clays
25
2,'
Fig. 3. Distribution of respirator~ activity between viable. @, and non-viable, O. bacteria at different net grov~th rates.
578
I. WALKERand M. D^vIEs
The possibility that "'dead" bacteria may be active must now be taken into account when using metabolic parameters such as ATP or dehydrogenase levels 0/¢eddle and Jenkins, 1971 ) for estimating viability. It is likely that the results of previous determinations of this type would need to be reassessed. There is also the potential for improving sewage disposal methods. At present much of the incoming waste at a sewage works is converted to biomass, which needs further treatment. Non-viable-active bacteria would permit the mineralization of waste without the production of excess biomass. Conversely if the yield of biomass needs to be maximized for food or energy resources the system would need to be controlled to minimize the numbers of non-viable-active bacteria. This work was supported by an S.R.C., C.A.S.E., award in collaboration with the Water Research Centre, Stevenage.
REFERENCES Banks C. J., Davies M., Walker I. and Ward R. D. (19761 Biological and physical characterisation of activated sludge: a comparative experimental study of ten different treatment plants. J. War. Pollut. Control Fed. 75, 492--509.
Banks C. J. and Walker I. (19771 Sonication of activat~ sludge floes and the recovery of their bacteria on solid media. J. gen. Microbiol. 9g, 363-368. Burchett M. E. and Tchobanoglous G. (19741 Fatalities for controlling the activated sludge process b~ mean cell residence time. d. War. Pollut. Control. Fed. 46, 973 979. Department of the Environment (19711 War. Polh, t. Re.s-. H.M.S.O., London. Garrett M. T. (1958) Hydraulic control of activated sludge growth rate. Sewage ind. Wastes. 30, 252-261 Jones G. L. (19731. Bacterial growth kinetics: measurement and significance in the activated sludge process. Water Res. 7, 1475-1492. Jones K. (19651. Small scale techniques in soy,age treatment research. J. Proc. Inst. Sew. Pur!/? 478 484. Neijssel O. M. and Tempest D. W. (19761 The role of energy-spilling reactions in the growth of Klehsiella aeroaenes, NCTC 418 in aerobic chemostat cultures..4rchs Microbiol. 110, 305- 311. Pike E. B., Carrington E. G. and Ashburner P.A. ~1972} An evaluation of procedures of enumerating bacteria in activated sludge. J. appl. Bact. 35. 309-321. Postgate J. R. and Hunter J. R. 11962) The stH~i,al of starved bacteria. J. 9en. Microbiol. 29, 233-263. Stephenson M. (19381 A cell free enzyme preparation obtained from bacteria. J. Biochem. 22, 605-614. Weddle C. L. and Jenkins D. (1971) The viability and activity of activated sludge. Water Res. 5. 621-640. Wooldridge W. R. and Standfast. A. F. B. (19361. The role of enzymes in activated sludge and .sewage oxidation. J. Biochem. 311, 1542-1543,
THE RELATIONSHIP BETWEEN VIABILITY A N D RESPIRATION RATE IN THE ACTIVATED SLUDGE PROCESS I. WAI,KER and M. DAVIES Department of Biology, University of York, Heslington, York YOI 5DD, England (Received 5 September 1976) Abstract- .The possibility that non-viable bacteria could be responsible for a proportion of the activity of an activated sludge, was discussed by Jones (1973). Following this, the work reported here was undertaken to observe the relationship between the respiratory activity and the unit viability of activated sludges cultured at various net growth rates. Both viable counts and respiration rates increased to maximum values at a growth rate of 0.1 days-~. However. it was found that the apparent respiration rate per viable cell was not constant, as reported by Weddle and Jenkins (1971). but that it increased at low growth rates. At a growth rate of 0.1 days-~ the observed activity was six times that expected from the observed viability. The results are explained by attributing part of the activity to non-viable bacteria.
INTRODUCTION
Work done by Postgate and Hunter (1962), using cultures of Aerobacter aerogenes grown in a chemostat with replacement times of up to 11 days, showed that only 37.8~o of the bacteria were demonstrably viable at very low growth rates. Weddle and Jenkins (1971} have shown that the numbers of viable ceils m g volatile suspended solids (unit viability) in activated sludge vanes with respect to the mean cell residence time of the system. Banks et al. (1976) found that the viability of sludges from ten different municipal activated sludge units ranged from 0.23 × 1()~ to 5.9 × 10 ~ cells g ~ of sludge (dry weight): this is approximately 2-60°/, viability. Jones (1973) suggested that the presence of nonviable but active bacteria could account for the d i s erepancy between the observed metabolic activity of a sludge and the lower value predicted from the numbers of bacteria shown to be present (Department of the Environment, 1971). The aim of the work reported here was to study the relationship between the activity and viability of sludges cultured at different growth rates, using a respirometer fitted with an oxygen electrode. The potential respiration rate (obtained when substrate is not limitingl was preferred as a measure of activity, to the rate in situ where substrate concentration is low. To allow for possible variations in floc structure each sample of sludge was exposed to a range of sonication periods before viability determinations wcre made. The sludge used was cultured from, and grown on. a domestic sewage in order to produce a naturally adapted population. MATERIALS AND METHODS
Culture system. The activated sludge unit consisted of a two tank aeration system of 3 I capacity, each tank contained a four inch stone air diffuser, and a 3 1 settlement
tank. The latter was a modified funnel fitted with an overflow weir and a rotating scraper chain to prevent sludge from accumulating on the sides (Jones, 1965). Sewage from the University's sewerage system was collected daily at 10.45am and settled for 18h before use. It was stored under refrigeration and fed continuously by peristaltic pump to the first aeration tank at a rate of 1.21 h - t ; sludge was recycled from the settlement tank at 0.8 I h *. The net growth rate of the sludge was controlled b) wasting from the aeration tank. as described by Garrett (1958). Calculation of the mean cell residence time is based on the assumption that the settlement tank contents has an average solids concentration equivalent to the mixed liquors solid's concentration (Burchett and Tchobanoglous, 1974). The volume of mixed liquor wasted each day from the aeration tank was calculated from the formula (~, + t'~ /"~ . . . . . . . 0r
F,.' .~'~
I X
(11
where: F~. = volume of mixed liquor to be removed each day, 1, X = concentration of solids (dry weightl in the mixed liquor, g. 1- ~, Va = volume of aeration system. 1. V, = volume of settlement tank. 1, F~ = tlow rate of effluent leaving the settlement tank, i.d-I X~ = concentration of solids (dry weight~ in the effluent. g.1-1. 0~ = required mean cell residence time Im.c.r.t.I, days, (equivalent to I/It where u = net growth rate, days- t). When F,. was less than 0.51 mixed liquor was wasted manually once a day; when F,,. was greater than 0.51 mixed liquor was automatically wasted by peristaltic pump once an hour. Viability determination. A five ml sample of mixed liquor was diluted ten-fold with sodium tripolyphosphate buffer (Pike et al., 1972), and placed in an ice cooled vessel. The sample was then sonicated using a Dawc Soniprobe (Model No. 1130) at a scale reading of 3.6. determined to be the optimum power output by Banks and Walker (1976). One ml samples were removed from the vessel 40, 60 and 80sec after commencement of sonication: the samples were serially diluted in polyphosphatc buffer and
575
I. WALKER and M. DAVIES
576
duplicate 0.1 ml vol of the 10 -~ and 10 " dilutions were spread on to agar plates. The medium used was C . G Y agar (casitone 5 g ' l - ' , glycerol 5 g . l '. yeast extract I g. 1- J, oxoid agar No. 3 13 g.l- ~) as recommended for maximal recovery by Pike et al. (19721. The colonies were counted after seven days incubation at 2 r C . Results are expressed as unit viability, that is the maximum viable cell count/g dry weight of mixed liquor suspended solids. Respiration rate determination. Oxygen uptake rates were measured in a Rank respirometer. 7 ml capacity, at 20C. Settled sewage was sampled from the holding tank, filtered through a "Millipore" membrane with a pore diameter of 0.45/~m and then aerated at room temperature for 10 rain, Five ml of this sewage was added to the incubation vessel and was allowed to equilibrate at 2 0 C after which 2 ml of the mixed liquor to be assayed was added: the vessel was sealed from the atmosphere and the fall in oxygen tension of the mixture was recorded. After 4-.Stain the incubation was stopped and 5 ml of the sample was withdrawn, the dry weight of this sample was determined. Four incubations were carried out for each mixed liquor assayed and the mean of these values is expressed as mg oxygen utilized g- ' of mixed liquor {dry weight) h- '. Each result reported is the mean of four determinations obtained when the activated sludge units were stable at the required growth rate. The units were classified as unstable if the C.O.D. of the sewage diverged by more than 20% from the mean of the previous three days. In all cases, excepting the lowest growth rate, the units had been operating at the maintained growth rate for at least one m.c.r.t. before measurements were recorded.
Table I. Concentration of mixed liquor suspended solids at different values of net growth rate 0,-~, days J
MLSS. mg.I :
0.0075 0.0480 0.(090 0.1520 O.1661) o. 1750 O.2OOO
7815 (~550 4() I 0 2825 245(I 28o0 232o 17(X)
0.3080 0.4090
[490 I l)l8
0.4330 1.0040
1260
2.7000
571)
It was found that both viable ceil counts a n d respiration rates increased with increasing values of 0; to reach a m a x i m u m value where 0¢- ~ = 0.7-O.~ d a y - ' (Fig. 1). Viable cell counts increased slightly at the lowest growth rates a n d then rose rapidly from 0.5 x 10 ~ cells g - ' to a m a x i m u m of 4.5 x 1('0 ~ cells g - ~. Respiration rates increased rapidly from t 3.2 mg O 2 g - ' h ' at the lowest net growth rate to a maxim u m of 70 mg 0 2 g - ~ h - ~. The relationship between net growth rate and thc a p p a r e n t respiration rate per viable cell counted, R a. is shown in Fig. 2. At the higher growth rates R, was constant at a value of 1.46 x 1 0 - ' : mg O , consumed per viable bacterium h - t . As the growth rate fell below 0.4 d a y - ' R, increased approximately six fold to a value of 9.5 × 10 -~-' mg 0 2 consumed per viable bacterium h - L where 0 [ ~ = 0.1 d a y ', and then fell toward zero,
RESULTS The m e a n C.O.D. of the sewage fed to the units on the days of experimentation was 290 nag. 1 - *, ranging from 260 to 3 1 5 m g ' l - L Effluent C.O.D, varied from 20 to 50 mg. 1-~ a n d effluent suspended solids varied from 10 to 3 0 r a g - I - ~ . Net growth rate, 0 [ ~, was calculated from a rearrangement of equation (1) a n d was 0,0075 day-~ at its lowest and 2.7 d a y at highest. The concentration of solids in the aeration system decreased with increasing values of 0,: Table 1.
D I S C U S S I O N
F r o m Fig. 1 it would appear that above a net growth rate of a r o u n d 0.9 d a y t the viable cell counts
b
60
L
¢3"
.c
°/ 50
•
,.c
(5 ~ 4 0
.
g C3 0.
30
20
J 0
4
>i
0
1 0
---
0.25
g --
I 0.50 Net
1
1
0.75 qrowth
J__l
J.O0 rate,
L25 8~',
i
,1 2.50
3.00
days-'
Fig. t. Effect of net growth rate on unit viability, O, and on respiration rate, 0.
Relationship between viability and respiration rate
577
'0 x
J~ x
6 v x c~ 0
4
O
1
I 0.25
I 0.50 Net
I
I
0.75 growth
I
I.O0
~ c I,
rote,
.
~.25
.
.
.
I
I
2.50
2 7O
c l o y s -I
Fig. 2. Effect of net growth rate on the apparent respiration rate per viable bacterium. Ra.
cannot be increased. This would indicate that the culture is I(X)~,~ viable and compares well with the data of Postgate and Hunter (1962) and Weddle and Jenkins (1971). Thc calculated dry weight of an average bacterium, at a net growth rate of 2.7 day- ~, of 2.22 x 10 ~-'g is not unreasonable if the culture is 10~)",, viable and the respiration rate of 1.46 x 1 0 - 1 2 m g O 2 bacterium -~ h t at the ~ m c net growth rate, is in good agreement with the rate obtained by Wcddlc and Jenkins (1971). 1.01 x 10 ~2 mg 02 bactcrium-~ h ~. Below a net growth of 0.9 day : the bacteria are probably unable to maintain their viability longer than the mean cell residence time. resulting in a proportion of the bacteria in the culture system being non-viable. If viable bacteria are rcsponsiblc for the total respiratory activity of a sludge then it is reasonable to expect that the observed respiration rate per viable cell should be constant over a wide rangc of net growth rates. This is a conclusion drawn by Weddle and Jenkins ( 1971 ). although their standard deviation is more than 60",, of their mean. Figure 2 contradicts this expectation, with viable bacteria appearing to respire more than six times faster at a net growth rate of 0.1 da 5- i. than at a net growth rate of 2.7 day-~. it is unlikely that this apparent increase in respiratory activity at lo~s. net growth rates is due to an increase in the maintenance requirements of the bacteria or to a "'slip" type of activity due to substrate limitations (Ncijssel and Tempest. 1976). These reactions cannot explain the fall in the apparent respiration rates at the lowest net growth rates. The most probable reason for the observations is that at the lower net growth rates a proportion of the respiratory activity can bc attributed to dying, non-viable bacteria. The process of bacterial death in slow growing continuous cultures is not clearly understood; Postgate's work (1962) has indicated that "dead" cells retain their osmotic integrity and several authors have reported that loss of viability is not necessarily associated with
loss of biochemical activity (Wooldridgc and Standfast, 1936; Stephenson, 1928). By assuming that the respiration rate of an average bacterium is 1.46 x 1 0 - ~ " m g 0 2 h ~ and that this value is independent of the net growth rate. the respiratory activity of the viable bacteria can be calculated: subtraction of this from the total activity should give an estimate of the activity due to non-viable bacteria. It can be seen in Fig. 3 that. at a growth rate of 0.1 d a y ~ over 80~o of the total respiration could bc attributed to non-viable bacteria. It is considered that the shape of Fig. 3 is duc to the death and decay rates of viable and non-viable bacteria increasing as the sludge net growth rate decreases. The total number of non-viable cells would increase but the proportion of these that arc active decreases. The changes are likely to bc due to increases in the maintenance requirement of the culture: as the solids level increases the total amount of substratc required to maintain all the cells in an active state increases, at some point the substratc supplied is insufficient to meet the needs of the culture and the rate of decay increases. .oo
o
80
o
./.
"6 6C
40
If o
0.2
0.4 Net
0.6
08
g r o w t h rate,
e
,O clays
25
2,'
Fig. 3. Distribution of respirator~ activity between viable. @, and non-viable, O. bacteria at different net grov~th rates.
578
I. WALKERand M. D^vIEs
The possibility that "'dead" bacteria may be active must now be taken into account when using metabolic parameters such as ATP or dehydrogenase levels 0/¢eddle and Jenkins, 1971 ) for estimating viability. It is likely that the results of previous determinations of this type would need to be reassessed. There is also the potential for improving sewage disposal methods. At present much of the incoming waste at a sewage works is converted to biomass, which needs further treatment. Non-viable-active bacteria would permit the mineralization of waste without the production of excess biomass. Conversely if the yield of biomass needs to be maximized for food or energy resources the system would need to be controlled to minimize the numbers of non-viable-active bacteria. This work was supported by an S.R.C., C.A.S.E., award in collaboration with the Water Research Centre, Stevenage.
REFERENCES Banks C. J., Davies M., Walker I. and Ward R. D. (19761 Biological and physical characterisation of activated sludge: a comparative experimental study of ten different treatment plants. J. War. Pollut. Control Fed. 75, 492--509.
Banks C. J. and Walker I. (19771 Sonication of activat~ sludge floes and the recovery of their bacteria on solid media. J. gen. Microbiol. 9g, 363-368. Burchett M. E. and Tchobanoglous G. (19741 Fatalities for controlling the activated sludge process b~ mean cell residence time. d. War. Pollut. Control. Fed. 46, 973 979. Department of the Environment (19711 War. Polh, t. Re.s-. H.M.S.O., London. Garrett M. T. (1958) Hydraulic control of activated sludge growth rate. Sewage ind. Wastes. 30, 252-261 Jones G. L. (19731. Bacterial growth kinetics: measurement and significance in the activated sludge process. Water Res. 7, 1475-1492. Jones K. (19651. Small scale techniques in soy,age treatment research. J. Proc. Inst. Sew. Pur!/? 478 484. Neijssel O. M. and Tempest D. W. (19761 The role of energy-spilling reactions in the growth of Klehsiella aeroaenes, NCTC 418 in aerobic chemostat cultures..4rchs Microbiol. 110, 305- 311. Pike E. B., Carrington E. G. and Ashburner P.A. ~1972} An evaluation of procedures of enumerating bacteria in activated sludge. J. appl. Bact. 35. 309-321. Postgate J. R. and Hunter J. R. 11962) The stH~i,al of starved bacteria. J. 9en. Microbiol. 29, 233-263. Stephenson M. (19381 A cell free enzyme preparation obtained from bacteria. J. Biochem. 22, 605-614. Weddle C. L. and Jenkins D. (1971) The viability and activity of activated sludge. Water Res. 5. 621-640. Wooldridge W. R. and Standfast. A. F. B. (19361. The role of enzymes in activated sludge and .sewage oxidation. J. Biochem. 311, 1542-1543,