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Population density and enzyme activities of heterotrophic bacteria in sewer biofilms and activated sludge
;Vat. Res. Vol. 28, No. 6, pp. 1341-1346, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/94 $7.00 + 0.00
Pergamon
POPULATION DENSITY A N D ENZYME ACTIVITIES OF HETEROTROPHIC BACTERIA IN SEWER BIOFILMS A N D ACTIVATED SLUDGE HILDE LEMMER*~, DORIS ROTH and MARGIT SCHADE Bavarian State Agency for Water Research (BayLWF), Kaulbachstr.37, D-80539 Munich, Germany
(First received May 1993; accepted in revisedform September 1993) Abstract--The heterotrophic activity of different wastewater biocenoses is assessed. The biofilms originate from a sewer discharging domestic wastewater and from a sewer discharging trade wastewater with chromium and nickel contamination. The activated sludge was taken from the aeration tank from the high load stage (F:M ratio 0.8kgkg -~ d -~) and from the low load stage (F:M ratio 0.1 kgkg -~ d -I) of a two-stage municipal wastewater treatment plant. We determined the population density of heterotrophic saprophytes, polymer degrading bacteria, ammonifying bacteria as well as of nitrate reducers and denitrifiers. Enzyme activities were determined as relative substrate turnover rates for esterase, ~t- and fl-glucosidase, for phosphatase and for L-alanine-aminopeptidase. The population density of heterotrophic saprophytes was one order of magnitude higher in the high load stage compared to the low load activated sludge. The sewer biofilms showed one to two orders of magnitude higher counts per g dry weight for the heterotrophic bacteria as compared to the activated sludges. The turnover rates of esterase and L-alanine-aminopeptidase were higher than those of ~- and fl-glucosidase and phosphatase. The sewer biofilms are highly active biocenoses both with respect to population densities as with respect to enzyme activities. Their heterotrophic activity is comparable with the one in high load activated sludge. Even the bacteria in the biocenosis which is exposed to high concentrations of chromium and nickel showed a high activity.
Key words--sewer biofilm, activated sludge, heterotrophic bacteria, polymer degrading bacteria, ammonifying bacteria, nitrate reducing bacteria, denitrifying bacteria, enzyme activities, heavy metals
INTRODUCTION
Biofilms in sewer systems have been investigated which are subject to chemical pollution with heavy metals ( G u t e k u n s t a n d H a h n , 1985) and chlorinated h y d r o c a r b o n s (Laschka a n d T r u m p p , 1991). As a result of a d s o r p t i o n processes these pollutants stick to the biofilm a n d can easily be m o n i t o r e d in order to identify polluters. Thus pollution of the wastewater a n d f u r t h e r m o r e c o n t a m i n a t i o n of waste sludges can be prevented. Waste sludges can then be used as a n agricultural fertilizer a n d need not be deposited in landfills. Biological investigations into the biocenosis of sewer biofilms a n d their activity are scarce. G u t e k u n s t (1989) reports some data on bacteria as well as o n p r o t o z o a a n d m e t a z o a sampled from biofilms of sewers discharging either little polluted municipal wastewater or industrial wastewater cont a m i n a t e d by heavy metals. In this study we focus on the investigation of the heterotrophic activity of the bacterial sewer biofilm biocenosis. W e determined two f u n d a m e n t a l ecological p a r a m e t e r s for the characterization o f a n ecosys-
*Author to whom all correspondence should be addressed.
tern: concentration of biomass, i.e. quantification of the density of various physiological groups of bacteria and determination of enzymatic t u r n o v e r rates of the biofilm biocenosis. D a t a from sewer biofilms with a n d without heavy metal c o n t a m i n a t i o n are c o m p a r e d with those gained from activated sludge from a two-stage municipal wastewater t r e a t m e n t plant.
MATERIALS AND METHODS
The sewer biofilms studied here originate either from a sewer discharging domestic wastewater ("domestic biofilm" = DB) or from a sewer discharging trade wastewater with chromium and nickel contamination ("trade biofilm" = TB). The thickness of the biofilms was in the range of 0.1-0.2 cm. Their texture was soft to granular. Samples were scraped or wiped off, stored in a sterile Petri dish and cooled for transport to the lab. It was not possible to take samples without affecting the biofilm layers. Thus in this study we could not yet determine population densities and activities separately for the different biofilm layers. All results refer to a mixed sample comprising the entire thickness of the biofilm. Activated sludge samples were taken from the aeration tanks from the high load stage (F: M ratio 0.8 kg kg-] d-~) and the low load stage (F:M ratio 0.1 k g k g - l d -I) of a two-stage municipal wastewater treatment plant.
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HILDE LEMMER et al.
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Sample preparation 1 g of the biofilm samples was suspended in 100ml tap water. Sodiumpyrophosphate in a concentration of 2.8 g 1- t was added to facilitate dispersion. The suspension was homogenized with a high speed cutting rod (Krups 3 mix) for 1 min and treated with ultrasonic for 2 min. Activated sludge samples were homogenized by an Ultraturrax treatment for 4 min on ice.
Quantification of physiological groups of bacteria The density of heterotrophic bacteria was assessed by surface plate count on TSA-medium [trypticase soy broth (BBL 11768) 30g1-1, bacto agar (Difco 0140-01) 1 5 g l - t ] . Samples of 0.1 ml from a 1: 10 dilution series were spread on duplicate agar plates and incubated at 22°C for 2 days. Colony forming units (cfu) were counted on plates of the series featuring approx. 30-300 cfu. The statistical error was in the range of 4-13%. The density of proteolytic bacteria was assessed by surface plate count on a gelatine medium as described by Pitt and Dey (1970). Polysaccharide degrading bacteria were counted on a starch medium as described by Reichardt (1978). The density of lipolytes was assessed on a medium containing olive oil according to Rhodes (1959) modified by Lemmer (1985). The density of ammonifying bacteria was determined with the membrane filter technique as described by Daubner and Ritter (1972). The concentration of nitrate reducers (NO 2 production) as well as the density of gas producing "true" denitrifiers ( N 2 0 and/or Nz production) were assessed by the most probable number (MPN) method as described by van Gylswyk (1961) in Tan and Overbeck (1973).
Determination of enzyme activities Enzyme activity was measured as described by Obst and Holzapfel-Pschorn (1988) with slight modifications. Esterase activity was assessed with the substrate fluorescein diacetate (Serva 21575), the activity of ~-glucosidase and fl-glucosidase with p-nitrophenyl-~-o-glycopyranoside (Serva 30716) and 4-nitrophenyl-fl-o-glycopyranoside, respectively. Phosphatase activity was determined with the substrate 4-nitrophenyl phosphate disodium salt (Merck 6850), aminopeptidase activity with e-alanine-4-nitroanilide hydrochloride (Merck 1014). RESULTS AND DISCUSSION
Heavy metal concentration in the sewer biofilms H e a v y m e t a l c o n c e n t r a t i o n in b o t h t h e biofilm from the sewer discharging domestic wastewater
( D B ) a n d t h e o n e d i s c h a r g i n g t r a d e w a s t e w a t e r (TB) w a s d e t e r m i n e d by I C P - O E S t h r e e t i m e s w i t h i n t h e i n v e s t i g a t i o n p e r i o d F e b r u a r y 1 9 9 2 - F e b r u a r y 1993. T h e c h r o m i u m c o n c e n t r a t i o n w a s in t h e r a n g e o f 1 6 6 0 - - 2 4 6 0 m g k g i d r y w e i g h t a n d nickel w a s in t h e r a n g e o f 8 4 0 - 1 2 5 0 m g kg -1 d r y w e i g h t in t h e T B - b i o cenosis. T h e c o n c e n t r a t i o n s o f t h e s e h e a v y m e t a l s were a b o u t t w o o r d e r s o f m a g n i t u d e lower in t h e DB-biocenosis.
Dry solid matter content T h e biofilm f r o m t h e sewer d i s c h a r g i n g d o m e s t i c w a s t e w a t e r w a s g r a n u l a r a n d w h i t i s h grey in color. T h e m e a n v a l u e o f t h e d r y solid m a t t e r w a s 0 . 1 4 g g J. T h e volatile f r a c t i o n varied f r o m 0.37 to 0.85 g g - t . T h e biofilm f r o m t h e sewer d i s c h a r g i n g t r a d e w a s t e w a t e r (TB) w a s soft, p a r t l y fibrous. T h e c o l o r w a s grey to r e d d i s h b r o w n . D r y solid m a t t e r w a s d e t e r m i n e d as 0.06 g g-~ w i t h a volatile f r a c t i o n o f 0 . 2 9 4 ) . 8 6 g g - t . T h e lower v a l u e s o f d r y solid m a t t e r are e x p l a i n e d by t h e s o f t e r s t r u c t u r e o f t h e T B c o m p a r e d to t h e D B . For the activated sludges from the high load stage the m i x e d l i q u o r s u s p e n d e d solids ( M L S S ) were in t h e r a n g e o f 1.7-3.2 g 1-~ w i t h a m e a n v a l u e o f 2 . 4 g 1- t , t h e volatile f r a c t i o n w a s a b o u t 7 5 % . F o r t h e activ a t e d s l u d g e s f r o m t h e low l o a d s t a g e t h e M L S S were d e t e r m i n e d as 2 . 9 - 1 1 . 4 g l ~ with a m e a n v a l u e o f 5.2 g l l, t h e volatile f r a c t i o n w a s a b o u t 6 0 % .
Population density of various heterotrophic bacteria Population densities of various physiological groups of heterotrophic bacteria originating from b o t h sewer biofilms a n d a c t i v a t e d s l u d g e s are s h o w n in T a b l e 1. T h e c o u n t s are related to d r y w e i g h t as well as to volatile d r y solid m a t t e r . T h e e r r o r b a r s listed refer to 1 cr e s t i m a t e s (n - 1).
Heterotrophic saprophytes Bacteria c o u n t s o f total h e t e r o t r o p h i c s a p r o p h y t e s in t h e biofilm o r i g i n a t i n g f r o m d o m e s t i c w a s t e w a t e r are in the r a n g e o f t h o s e f o u n d in a c t i v a t e d s l u d g e
Table 1. Population densities of various physiological groups of heterotrophic bacteria originating from both sewer biofilms and activated sludges, given as colony forming units (cfu) and most probable number (MPN) related to g dry weight (DW) and volatile dry solid matter (vDW), respectively. The error bars listed refer to 1 a estimates (n - 1) Biofilm from domestic wastewater (n=5-10) Heterotrophic bacteria Proteolytic bacteria
cfu cfu cfu cfu Polysaccharide cfu degrading bacteria cfu Lipolytic bacteria cfu Ammonifying bacteria Nitrate reducing bacteria Denitrifying bacteria
per per per per per per per
10I° 7.3 x 101°+3.3 x 10~° 10~° 1.3 × 10~+6.3 x 10~° l09 2.5 x 10t° + 5.6 × 10t° 109 3.2x 10~°+ 6.7 × 10~° 109 2.1 x 10~°+ 1.5 x 10~° 10t° 3.3 × 10~°+ 2.1 × 10~° 109 2.0 × 10t° _+2.8 x 10]° cfu per g vDW 5 . 3 x 1 0 9 5.1 x l09 2.5 × 10~°+3.3 × 10I° cfupergDW l.l×10t°__l.3×10 t° 1.4×10H+1.7×10 tl cfupergvDW 1.8×10~°+2.2×10 ~° 2 . 6 x l O H + 3 . 5 × I 0 H MPN per g DW 2.8 × 10tl +3.6 × 10H 8.5 × 1011+8.1 × 10H MPN per g vDW 4.4× 10H___6.4 × 10II 1.4x I012+1.3 × 10~2 MPN per g DW 3.0× 10]°_+2.7x 10r° 3.0× 10~-+3.6 × 10H M P N p e r g v D W 4 . 3 × 1 0 t ° + 3 . 0 x 1 0 ~° 4.3xlOH_+5.1×I0 ~t
*Single determination; n = 1.
g g g g g g g
DW vDW DW vDW DW vDW DW
1.3 × 10t°~ 1.3x 2.4 x 10~°_+2.4 x 3.7 x 109 + 5.0 × 5.5x 109_+6.6× 8.1 x 1 0 9 7.2 × 1.3 x 10~°___1.2 x 2.7 x 109 + 1.8 ×
Biofilm from trade wastewater (n =5 10)
High load activated sludge (n= 10-15) 2.7× 10t°+ 1.1 × 10t° 3.8 × 10t°_+ 1.6 x 10]° 1.6 × 109* 2.2x 109 8.1 × 10~°* I.I × 10II 1.4 × 10~°* 1.9x 10~° 1.3 ×10~° _+8.6 ×109 1.7 ×10~° -+1.2 ×10 t° 3.0 × 10~°_+2.2 × 10I° 4.0× 101°+2.9 × 10I° 3.1 × 109+4.8× 109 4.0 x l09 -+ 6.3 ×109
Low load activated sludge (n =10-15) 1.8 x 109± 1.7 × 109 3.3×109_+3.0×109
6.5 × l0 s* 1.2× 10~ 5.9 x 103* 1.1 × 10I° 6.5 x 10~* 1.2×109 5.1 ×10s_+3.1xl0 s 9.5×10s+6.3×10 s 2.6 × 109±2.5 x 109 4.7× 109--+4.6× 109 2.3× 10s-+2.3 x l0 s 4.3×10~+4.2×10 s
Sewer biofilms and activated sludge from the high load stage. The counts in the biofilm from trade wastewater are one order of magnitude higher. The lowest counts were found in the low load stage. Gutekunst (1989) reported a density of heterotrophic bacteria of 109 cfu g ~volatile dry solid matter in both sewer biofilms originating from domestic wastewater and biofilms from heavy metal contaminated wastewater. These counts are quite low compared to our results. In contrast to our results in the investigations of Gutekunst the count of heterotrophs did not differ between the domestic wastewater biofilm and the biofilm from industrial wastewater. However, the author found a 10-fold higher population density of aeromonads, pseudomonads and clostridia in the domestic sewer compared to the industrial one. In activated sludge the density of heterotrophic bacteria was determined by Prakasam and Dondero (1967) as 2.6x 108cfuml -~, i.e.--related to dry weight--in the same range as those found in the high load stage of the municipal plant investigated here. The counts reported by G/ide (1982) were in the same order of magnitude (108 cfu ml-~). Benedict and Carlson (1971) found densities of heterotrophic bacteria of 2 x 10~1-5 x 10Hcfug -t dry weight in the activated sludge of a municipal sewage plant. Schmider and Ottow (1986) found bacterial numbers between 2 x 106 and 2 x 107 MPN ml i for heterotrophic bacteria from the aeration tank of a low load municipal plant. Wetzel and Dott (1984) reported average counts of heterotrophic bacteria of 4.8 x 108 cfu mlin activated sludge from the high load stage (F:M ratio 4.3 kg kg i d- 1) and of 4.8 × l07 cfu ml-l in the low load stage (F:M ratio 0.13kgkg 1d-l) of a two-stage municipal plant treating wastewater with 40% domestic and 60% trade/industrial wastewater. The MLSS were determined as 2.8 and 3.5gl ~, respectively. Thus the counts translate to 1.7 x 10H and 1.4 x 101°cfu g 1 dry weight. The counts in the high load and in the low load sludge differ in one order of magnitude as did the counts in the present study. K~impfer (1988) found a density of heterotrophic bacteria of 1 x 107-9 x 107cfuml -~ in the activated sludge of a one-stage plant (F:M ratio 0.25 kg kg-~ d-~; MLSS 3.3gl ~), i.e. 3 x 109-2.7 × 10J°cfug ~ dry weight. These values agree with the values we found in the activated sludge of the two-stage municipal plant.
Polymer degrading bacteria: proteolytic bacteria, polysaccharide degrading bacteria and lipolytic bacteria The counts of polymer degrading bacteria were in the range of 109-101° in both biofilm biocenoses and the high load activated sludge. Those in the low load sludge were one order of magnitude lower, similar to those of heterotrophic bacteria. Literature data on the densities of proteolytic and lipolytic as well as on polysaccharid degrading bac-
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teria are scarce. Dott (1980) reported on heterotrophic bacteria populations in slime originating from drinking water systems. The counts for gelatinase-positive bacteria were in the range of 10-20% of the count of total heterotrophs. Dott and Wetzel (1984) found higher counts of "polymer degrading" bacteria in the sludge from the high load stage of a two-stage wastewater treatment plant compared to the low load sludge. This is in agreement with our results. The authors stress that the bacteria from the high load milieu are physiologically more flexible, many of them are able to degrade polymers. Gfide (1982) found that the total counts of "lytic bacteria" (i.e. lysis of polysaccharides such as cellulose, chitin, dextran, pectin and xylan and lysis of gelatine) in wastewater biocenoses are always at least one order of magnitude lower than those of heterotrophs assessed on casitone-yeast agar.
Bacteria of the nitrogen cycle: ammonifiers, nitrate reducers and denitrifiers This group developed high population densities in both the DB- and the TB-biocenosis. They were in the same range or even higher than in the high load activated sludge. In the low load stage average counts of ammonifiers were quite low. The counts of gas forming "true denitrifiers" in both activated sludge samples were about one order of magnitude lower than the counts of nitrate reducers. Compared to the densities found in the biofilm biocenoses the counts in the low load sludge are two to three orders of magnitude lower. The high oxygen concentration as well as the limitation of carbon sources in the low load stage do not support denitrification processes. In "classical denitrification" the enzyme system is induced by anoxic conditions only. In contrast, aerobic denitrification with a constitutive enzyme system was observed only in a few cases (Robertson and Kuenen, 1989). This could be a reason for the low density of the heterotrophic denitrifying population we found in the low load activated sludge community. Schmider and Ottow (1986) investigated the bacteria populations in activated sludge from a low load wastewater treatment plant. In the aeration tank they found between 2 x 106 and 2 x 107MPNml ~ of heterotrophic bacteria, the density of (gas producing) denitrifiers was in the range of 5 x 105 and 1.1 x 107 MPN ml -~. The MPN-assessment of denitrifying bacteria is methodologically problematic. The inoculum in the test tube is a mixed culture of assimilatory and dissimilatory nitrate reducers and "true denitrifiers" as well as aerobic denitrifiers, all competing for the nitrate. "True denitrifiers" may be occasionally outcompeted. This leads to "false negative" results with "improbable combinations of positive and negative results", if gas production is chosen as a test criterium.
H1LDE LEMMER et al.
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Table 2. Enzyme activities of various physiological groups of heterotrophic bacteria from sewer biofilms and activated sludges, given as % substrate turnover (a) and as mU (b) related to mg dry weight (DW) and mg volatile dry solid matter (vDW), respectively. The error bars listed refer to I cr estimates (n - 1)
(a)
Esterase cc-Glucosidase /~-Glucosidase Phosphatase L-Ala-aminopeptidase
% 0/° % 0/° % % % % % %
turnover turnover turnover turnover turnover turnover turnover turnover turnover turnover
per per per per per per per per per per
h h h h h h h h h h
and and and and and and and and and and
mg mg mg mg mg mg mg mg mg mg
DW vDW DW vDW DW vDW DW vDW DW vDW
Biofilm from domestic wastewater (n = 5-10)
Biofilm from trade wastewater (n ~ 5-10)
High load activated sludge (n = 5-10)
Low load activated sludge (n = 5-10)
42.3 + 21.0 69.3 4- 44.0 4.5 4- 2.5 7.0 4- 2.6 5.3 _+ 1.6 9.1 4-4.8 18.1 4- 8.8 29.3 4- 10.5 34.5 4- 24.2 53.4_+28.0
95.6 4- 50.8 136.7 _+ 79.4 9.4 + 4.8 12.4 4- 6.4 14.0 4- 11.0 18.24- 13.2 32.1 ___16.5 43.4 4- 25.5 93.1 4- 47.2 123.44-63.4
51.1 4- 14.6 67.8 4- 18.8 6.3 4- 1.5 8.4 4- 2.0 4.6 + 0.9 6.2+ 1.2 17.9 4- 3.2 24.0 4- 4.2 113.7 4- 34.6 152.04-46.7
21.0 4- 5.8 37.1 4- 8.4 1.6 4- 0.3 3.0 4- 0.5 1.5 4- 0.3 2.84-0.6 6.3 + 1.5 11.5 4- 2.8 39.1 4- 3.6 71.24-7.7
Biofilm from domestic wastewater (n = 5-10)
Biofilm from trade wastewater (n = 5-10)
High load activated sludge (n = 5-10)
Low load activated sludge (n = 5-10)
1.4 + 0.7 22 ± 1.4 2.5 4- 1.4 3.9 4- 1.4 2.9 +_0.9 5.04-2.7 8.1 4- 4.0 13.2 4- 4.7 23.4 4- 16.4 36.24- 1 9 . 0
3.1 4- 1.6 4.4 4- 2.5 5.2 + 2.7 6.9 + 3.6 7.7 _+ 6.1 I0.1 4-7.3 14.4 4- 7.4 19.5 4- 11.4 63.2 4- 32.0 83.74-43.0
1.6 4- 0.5 2.2 4- 0.6 3.5 4- 0.8 4.7 + 1. t 2.6 4- 0.5 3.44-0.7 8.1 4- 1.5 10.8 4- 1.9 77.1 4- 23.5 103.1 4-31.7
0.7 4- 0.2 1.2 4- 0.3 0.9 4- 0.2 1.7 4- 0.3 0.9 4- 0.2 1.64-0.3 2.8 + 0.7 5.2 4- 1.2 26.5 4- 2.4 48.34-5.3
(h)
Esterase • -Glucosidase /~-Glucosidase Phosphatase L-Ala-aminopeptidase
mU mU mU mU mU mU mU mU mU mU
per per per per per per per per per per
mg mg mg mg mg mg mg mg mg mg
DW vDW DW vDW DW vDW DW vDW DW vDW
Enzyme activity of various populations of heterotrophic bacteria Enzyme activities of various physiological groups of heterotrophic bacteria from both sewer biofilms and activated sludges are shown in Table 2. In Table 2(a) the activities are given as % substrate turnover h -1, in Table 2(b) as mU. The values are related to mg dry weight and mg volatile dry solid matter, respectively.
Esterase activity Esterases are unspecific enzymes involved in the degradation of polymers. Esterase activity was determined in order to assess the total heterotrophic degradation activity of sewer biofilms and activated sludges. In the TB-biocenosis about 2-fold higher activities were found compared to the DB-biocenosis. This is in contrast to results of Gutekunst (1989) who found significantly higher activities of dehydrogenase and oxygen consumption in domestic sewer biofilms compared to industrial ones. The esterase activity of the activated sludge biocenosis from the high load stage was in the same range as the activity of the biofilm in the domestic sewer. Low load activated sludge was only half as active as the forementioned biocenoses. Esterase activities in biofilm bioeenoses from the sediment of the river Neckar/FRG were determined by Altmeier and Schweisfurth (1989) as 1-5/~ U mgdry weight. The activities of wastewater biocenoses
studied in the present work were three orders of magnitude higher.
Activity of ~-glucosidase and t~-glucosidase Glucosidases hydrolyse disaccharids originating from the degradation of polysaccharids, cc-Glucosidase cleaves the disaccharides maltose and saccharose. //-Glucosidase as a part of the cellulolytic enzyme system cleaves cellobiose. The activity in the TB-biocenosis was 2-fold higher, the one in the high load activated sludge in the same range as in the DB-biocenosis. The low load activated sludge showed a low activity, about a third of the DB-activity. A similar activity pattern was determined for the/~-glucosidase. Teuber and Brodisch (1977) reported glucosidase activities in activated sludge from three one-stage wastewater treatment plants in the range of 5.2-16.0 mU mg -~ protein. Assuming a protein content of about 50% of the dried solid matter (N/iveke and Tepper, 1979), the glucosidase activities are between 2.6 and 8.0mU/mg -j dry weight, i.e. compared to the activities related to volatile dry solid matter, in the same range as those found in this study for the biofilm biocenoses. Verstraete et al. (1976) found the saccharase activity to be specifically related to the organic matter content, i.e. organic pollutants such as proteins and carbohydrates. Random samples taken from the wastewater revealed somewhat higher COD values in the trade wastewater than in the
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Sewer biofilms and activated sludge domestic one. High maximum values suggest shock load conditions in the trade sewer compared to the domestic sewer.
decreased to 20% after 4 days of starvation. Thus the low values found by Nybroe et al. (1992) might be attributed to starvation effects induced by the low load of the plants they investigated.
Phosphatase activity
Phosphatases are esterases cleaving phosphomonoesters. They have a low substrate specifity and describe the total metabolic activity of a biocenosis. The activities of phosphatase found in the biofilm and activated sludge biocenoses show a similar pattern to the glucosidases. The activities found in the DB-biocenosis and the high load activated sludge are in the same range, the TB-biocenosis had a 2-fold higher activity. The low load activated sludge biocenosis was only a third as active as the organisms of the high load stage. Phosphatase activity is known to be related to eutrophication. The higher the degree of eutrophy the higher phosphatase activities were found (Hantke and Melzer, 1991). Verstraete et al. (1976) found heavy loads of organic pollutants in general and proteinaceous compounds in particular to induce increased phosphatase activity. Activity of L-alanine-aminopeptidase
Protein metabolism plays an important role in microbial communities, especially in the degradation of decaying biomass. Peptides are cleaved by aminopeptidases. These exopeptidases are specific for different amino acids, e.g. for alanine. The activity pattern of L-alanine-aminopeptidase in the biofilm and activated sludge biocenoses differed from that determined for the glucosidases and phosphatases. The activity in the DB-biocenosis was in the same range as in the low load activated sludge biocenosis. Both the TB-biocenosis and the high load activated sludge, however, showed a 3-fold higher activity. Teuber and Brodisch (1977) reported peptidase activities in three activated sludges in the range of 28.2, 55.3-76.3 m U m g -1 protein. Assuming a protein content of 50% in the dry matter of biomass (N/iveke and Tepper, 1979), we get around 14.1, 27.7 and 38.2 mU m g -1 dry weight. Brodisch et al. (1979) found a peptidase activity of 5 mU mg ~ dry weight in the return sludge from a wastewater treatment plant with a F: M ratio of about 0.2 kg kg-~ d - l . This activity is quite low compared to the activities in the biocenoses studied here. Nybroe et al. (1992) found for low load activated sludge from plants in Denmark that the peptidases show the highest substrate production rates of the enzymes investigated here. But the activity ofesterase and the glucosidases they report are lower than the values we found. However, the characteristics of the plants Nybroe et al. investigated are somewhat different. Generally we found enzyme activities higher than the values reported in the literature. Teuber and Brodisch (1977) found that specific enzyme activities
CONCLUSIONS
The population density of heterotrophic saprophytes we found in high load and low load activated sludge is similar to that reported in various other publications. The high load in the first stage of a two-stage wastewater treatment plant yielded one order of magnitude higher counts of heterotrophic bacteria compared to the low load second stage. Biofilms from sewers discharging domestic and trade wastewater revealed one to two orders of magnitude higher counts for the heterotrophic bacteria compared to high load and low load activated sludges. Enzyme activities show similar patterns in the substrate turnover rates of esterase, ct- and fl-glucosidase and phosphatase in the biofilm biocenoses as in activated sludge. However, the turnover rate of L-alanine-aminopeptidase is very high in the high load activated sludge. Sewer biofilms are fixed film systems. Their ability to efficiently remove carbon and nitrogen from wastewater is well documented. For many years the most prevalent applications of these systems are carbon and nitrogen removal (Bryers and Characklis, 1990). We found population densities and enzyme activities which reveal the sewer biofilms to be a highly active biocenosis. Their heterotrophic activity is comparable with the one in high load activated sludge or even exceeds it. Compared to suspended microorganisms those attached to biofilms are known to be more resistant to toxic materials such as heavy metals. The high activity we found for the bacteria in the TB-biocenosis exposed to high concentrations of chromium and nickel confirms this statement. We find highly developed eukaryotic organisms such as slime molds and a variety of proto- and metazoa to be abundant in the DB-biocenosis. In the TB-biocenosis, on the other hand, they occur only in low numbers or else are even lacking. Eukaryotic organisms, in contrast to bacteria, seem to be much more susceptible to pollution by heavy metals. The combination of determining both population densities and enzyme activities separately for specialized groups of bacteria turned out a useful and workable tool to describe the activity of a biocenosis. For biology to efficiently support the development and performance of wastewater treatment, more data of that kind are needed for prokaryotic as well as eukaryotic organisms from various wastewater biocenoses such as activated sludge from stabilization tanks, trickling filter biofilms, fluidized bed systems or biomass support particle systems.
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HILDELI~MMERet al.
Acknowledgements--We wish to thank Dagmar Laschka and Christine G~inther from the BayLWF for the heavy metal analyses by ICP--OES. We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft (Biofilm Project, Grant Le 724/1-1).
REFERENCES
Altmeier U. and Schweisfurth R, (1989) Untersuchungen zur mikrobiellen Charakterisierung yon uferfiltriertem Grundwasser. Vom. Wass. 73, 333-444. Benedict R. G. and Carlson D. A. (1971) Aerobic heterotrophic bacteria in activated sludge. Wat. Res. 5, 1023-1030. Brodisch K., Teuber M. and Hegemann W. (1979) Enzymaktivit/iten als Kennwerte des belebten Schlammes. GWF 120, 524-527. Bryers J. D. and Characklis W. G. (1990) Biofilms in water and wastewater treatment. In Biofilms (Edited by Characklis W. G. and Marshall K. C.), pp. 671-696. Wiley, New York. Daubner I. and Ritter R. (1972) Zur Methodik des quantitativen Nachweises yon ammonifizierenden und denitrifizierenden Bakterien im Wasser mit Anwendungsbeispielen (Baggersee-Untersuchungen). Int. Rev. ges. Hydrobiol. 57, 517-522. Dott W. (1980) Qualitative und quantitative Bestimmung von Bakterienpopulationen aus aquatischen Biotopen: 1. Mitteilung: Selektionierung yon Bakteriengruppen durch Isolierung auf verschiedenen N~ihrb6den. Zbl. Bakt. LAbt. Orig. B 170, 93-98. Dott W. and Wetzel A. (1984) Mikrobiologische Untersuchungen einer zweistufigen Adsorptionsbelebungsanlage: 2. Mitteilung: ln-vitro-Aktivithten der isolierten Bakterien. Z. Wass.-Abwass.-Forsch. 17, 182-185. Giide (1982) Interactions between floc-forming and non floc-forming bacterial populations from activated sludge. Curr. MicrobioL 7, 347-350. Gutekunst B. (1989) Praktische Erfahrungen und Ergebnisse aus Sielhautuntersuchungen zur Ermittlung schwermetallhaltiger Einleitungen. Korr. Abwass. 36, 1367-1375. Gutekunst B. and Hahn H. H. (1985) Schwermetalle in Sielh/iuten--Eine Mfglichkeit zum Nachweis von Einleitungen schwermetallhaltigen Abwassers in die Kanalisation. Vom. Wass. 65, 127-137.
Hantke B. and Melzer A. (1991) Phosphataseaktivit/it in drei Seen unterschiedlichen Trophiegrades. Verh. Ges. OkoL (Freising-Weihenstephan 1990). 20, 513-520. K/impfer P. (1988) Automatisierte Charakterisierung mikrobieller Lebensgemeinschaften. Hyg. Berl. 1, 1-248. Laschka D. and Trumpp M. (1991 ) Sielhautuntersuchungen zur Lokalisierung von AOX-Emittenten im Kanalnetz. Korr. Abwass. 38, 495-496. Lemmer H. (1985) Mikrobiologische Untersuchungen zur Bildung yon Schwimmschlamm auf Kl/iranlagen. Ph.D. thesis, Technical University Munich, Germany, N/iveke R. and Teppcr K.-P. (1979) Einfiihrung in die Mikrobiologischen Arbeitsmethoden mit Praktikumsaufgaben. Fischer, Stuttgart. Nybroe O., Jorgensen P. E, and Henze M. (1992) Enzyme activities in wastewater and activated sludge. Wat. Res. 26, 579-584. Obst U. and Holzapfel-Pschorn A. (1988) Enzymatische Tests fiir die Wasseranalytik. Oldenbourg, Miinchen. Pitt T. L. and Dey D. (1970) A method for detection of gelatinase production by bacteria. J. appl. Bact. 33, 687-691. Prakasam T. B. S. and Dondero N. C. (1967) Aerobic heterotrophic bacterial populations of sewage and activated sludge. I. Enumeration. Appl. Microbiol. 15, 461-467. Reichardt W. (1978) Einfiihrung in die Methoden der Gewiissermikrobiologie. Fischer, Stuttgart. Rhodes M. E. (1959) The characterization of Pseudomonas fluorescens. J. gen. Microbiol. 21, 221-263. Robertson L. A. and Kuenen J. G. (1983) Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium. J. gen. Microbiol. 129, 2847-2855. Schmider F. and Ottow J. C. G. (1986) Charakterisierung der denitrifizierenden Mikroflora in den verschiedenen Reinigungsstufen einer biologischen Kl/iranlage. Arch. Hydrobiol. 106, 497-512. Tan T. L. and Overbeck J. (1973) Okologische Untersuchungen iiber nitratreduzierende Bakterien im Wasser des PluBsees (Schleswig-Holstein). Z. Allg. Mikrobiol. 13, 71-82. Teuber M. and Brodisch K, E. U. (1977) Enzymatic activities of activated sludge. Eur. J. appl. Microbiol. 4, 185-194. Verstraete W., Voets J. P. and van Lancker P. (1976) Evaluation of some enzymatic methods to measure the bio-activity of aquatic environments. Hydrobiologia 49, 257-266.
Pergamon
POPULATION DENSITY A N D ENZYME ACTIVITIES OF HETEROTROPHIC BACTERIA IN SEWER BIOFILMS A N D ACTIVATED SLUDGE HILDE LEMMER*~, DORIS ROTH and MARGIT SCHADE Bavarian State Agency for Water Research (BayLWF), Kaulbachstr.37, D-80539 Munich, Germany
(First received May 1993; accepted in revisedform September 1993) Abstract--The heterotrophic activity of different wastewater biocenoses is assessed. The biofilms originate from a sewer discharging domestic wastewater and from a sewer discharging trade wastewater with chromium and nickel contamination. The activated sludge was taken from the aeration tank from the high load stage (F:M ratio 0.8kgkg -~ d -~) and from the low load stage (F:M ratio 0.1 kgkg -~ d -I) of a two-stage municipal wastewater treatment plant. We determined the population density of heterotrophic saprophytes, polymer degrading bacteria, ammonifying bacteria as well as of nitrate reducers and denitrifiers. Enzyme activities were determined as relative substrate turnover rates for esterase, ~t- and fl-glucosidase, for phosphatase and for L-alanine-aminopeptidase. The population density of heterotrophic saprophytes was one order of magnitude higher in the high load stage compared to the low load activated sludge. The sewer biofilms showed one to two orders of magnitude higher counts per g dry weight for the heterotrophic bacteria as compared to the activated sludges. The turnover rates of esterase and L-alanine-aminopeptidase were higher than those of ~- and fl-glucosidase and phosphatase. The sewer biofilms are highly active biocenoses both with respect to population densities as with respect to enzyme activities. Their heterotrophic activity is comparable with the one in high load activated sludge. Even the bacteria in the biocenosis which is exposed to high concentrations of chromium and nickel showed a high activity.
Key words--sewer biofilm, activated sludge, heterotrophic bacteria, polymer degrading bacteria, ammonifying bacteria, nitrate reducing bacteria, denitrifying bacteria, enzyme activities, heavy metals
INTRODUCTION
Biofilms in sewer systems have been investigated which are subject to chemical pollution with heavy metals ( G u t e k u n s t a n d H a h n , 1985) and chlorinated h y d r o c a r b o n s (Laschka a n d T r u m p p , 1991). As a result of a d s o r p t i o n processes these pollutants stick to the biofilm a n d can easily be m o n i t o r e d in order to identify polluters. Thus pollution of the wastewater a n d f u r t h e r m o r e c o n t a m i n a t i o n of waste sludges can be prevented. Waste sludges can then be used as a n agricultural fertilizer a n d need not be deposited in landfills. Biological investigations into the biocenosis of sewer biofilms a n d their activity are scarce. G u t e k u n s t (1989) reports some data on bacteria as well as o n p r o t o z o a a n d m e t a z o a sampled from biofilms of sewers discharging either little polluted municipal wastewater or industrial wastewater cont a m i n a t e d by heavy metals. In this study we focus on the investigation of the heterotrophic activity of the bacterial sewer biofilm biocenosis. W e determined two f u n d a m e n t a l ecological p a r a m e t e r s for the characterization o f a n ecosys-
*Author to whom all correspondence should be addressed.
tern: concentration of biomass, i.e. quantification of the density of various physiological groups of bacteria and determination of enzymatic t u r n o v e r rates of the biofilm biocenosis. D a t a from sewer biofilms with a n d without heavy metal c o n t a m i n a t i o n are c o m p a r e d with those gained from activated sludge from a two-stage municipal wastewater t r e a t m e n t plant.
MATERIALS AND METHODS
The sewer biofilms studied here originate either from a sewer discharging domestic wastewater ("domestic biofilm" = DB) or from a sewer discharging trade wastewater with chromium and nickel contamination ("trade biofilm" = TB). The thickness of the biofilms was in the range of 0.1-0.2 cm. Their texture was soft to granular. Samples were scraped or wiped off, stored in a sterile Petri dish and cooled for transport to the lab. It was not possible to take samples without affecting the biofilm layers. Thus in this study we could not yet determine population densities and activities separately for the different biofilm layers. All results refer to a mixed sample comprising the entire thickness of the biofilm. Activated sludge samples were taken from the aeration tanks from the high load stage (F: M ratio 0.8 kg kg-] d-~) and the low load stage (F:M ratio 0.1 k g k g - l d -I) of a two-stage municipal wastewater treatment plant.
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HILDE LEMMER et al.
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Sample preparation 1 g of the biofilm samples was suspended in 100ml tap water. Sodiumpyrophosphate in a concentration of 2.8 g 1- t was added to facilitate dispersion. The suspension was homogenized with a high speed cutting rod (Krups 3 mix) for 1 min and treated with ultrasonic for 2 min. Activated sludge samples were homogenized by an Ultraturrax treatment for 4 min on ice.
Quantification of physiological groups of bacteria The density of heterotrophic bacteria was assessed by surface plate count on TSA-medium [trypticase soy broth (BBL 11768) 30g1-1, bacto agar (Difco 0140-01) 1 5 g l - t ] . Samples of 0.1 ml from a 1: 10 dilution series were spread on duplicate agar plates and incubated at 22°C for 2 days. Colony forming units (cfu) were counted on plates of the series featuring approx. 30-300 cfu. The statistical error was in the range of 4-13%. The density of proteolytic bacteria was assessed by surface plate count on a gelatine medium as described by Pitt and Dey (1970). Polysaccharide degrading bacteria were counted on a starch medium as described by Reichardt (1978). The density of lipolytes was assessed on a medium containing olive oil according to Rhodes (1959) modified by Lemmer (1985). The density of ammonifying bacteria was determined with the membrane filter technique as described by Daubner and Ritter (1972). The concentration of nitrate reducers (NO 2 production) as well as the density of gas producing "true" denitrifiers ( N 2 0 and/or Nz production) were assessed by the most probable number (MPN) method as described by van Gylswyk (1961) in Tan and Overbeck (1973).
Determination of enzyme activities Enzyme activity was measured as described by Obst and Holzapfel-Pschorn (1988) with slight modifications. Esterase activity was assessed with the substrate fluorescein diacetate (Serva 21575), the activity of ~-glucosidase and fl-glucosidase with p-nitrophenyl-~-o-glycopyranoside (Serva 30716) and 4-nitrophenyl-fl-o-glycopyranoside, respectively. Phosphatase activity was determined with the substrate 4-nitrophenyl phosphate disodium salt (Merck 6850), aminopeptidase activity with e-alanine-4-nitroanilide hydrochloride (Merck 1014). RESULTS AND DISCUSSION
Heavy metal concentration in the sewer biofilms H e a v y m e t a l c o n c e n t r a t i o n in b o t h t h e biofilm from the sewer discharging domestic wastewater
( D B ) a n d t h e o n e d i s c h a r g i n g t r a d e w a s t e w a t e r (TB) w a s d e t e r m i n e d by I C P - O E S t h r e e t i m e s w i t h i n t h e i n v e s t i g a t i o n p e r i o d F e b r u a r y 1 9 9 2 - F e b r u a r y 1993. T h e c h r o m i u m c o n c e n t r a t i o n w a s in t h e r a n g e o f 1 6 6 0 - - 2 4 6 0 m g k g i d r y w e i g h t a n d nickel w a s in t h e r a n g e o f 8 4 0 - 1 2 5 0 m g kg -1 d r y w e i g h t in t h e T B - b i o cenosis. T h e c o n c e n t r a t i o n s o f t h e s e h e a v y m e t a l s were a b o u t t w o o r d e r s o f m a g n i t u d e lower in t h e DB-biocenosis.
Dry solid matter content T h e biofilm f r o m t h e sewer d i s c h a r g i n g d o m e s t i c w a s t e w a t e r w a s g r a n u l a r a n d w h i t i s h grey in color. T h e m e a n v a l u e o f t h e d r y solid m a t t e r w a s 0 . 1 4 g g J. T h e volatile f r a c t i o n varied f r o m 0.37 to 0.85 g g - t . T h e biofilm f r o m t h e sewer d i s c h a r g i n g t r a d e w a s t e w a t e r (TB) w a s soft, p a r t l y fibrous. T h e c o l o r w a s grey to r e d d i s h b r o w n . D r y solid m a t t e r w a s d e t e r m i n e d as 0.06 g g-~ w i t h a volatile f r a c t i o n o f 0 . 2 9 4 ) . 8 6 g g - t . T h e lower v a l u e s o f d r y solid m a t t e r are e x p l a i n e d by t h e s o f t e r s t r u c t u r e o f t h e T B c o m p a r e d to t h e D B . For the activated sludges from the high load stage the m i x e d l i q u o r s u s p e n d e d solids ( M L S S ) were in t h e r a n g e o f 1.7-3.2 g 1-~ w i t h a m e a n v a l u e o f 2 . 4 g 1- t , t h e volatile f r a c t i o n w a s a b o u t 7 5 % . F o r t h e activ a t e d s l u d g e s f r o m t h e low l o a d s t a g e t h e M L S S were d e t e r m i n e d as 2 . 9 - 1 1 . 4 g l ~ with a m e a n v a l u e o f 5.2 g l l, t h e volatile f r a c t i o n w a s a b o u t 6 0 % .
Population density of various heterotrophic bacteria Population densities of various physiological groups of heterotrophic bacteria originating from b o t h sewer biofilms a n d a c t i v a t e d s l u d g e s are s h o w n in T a b l e 1. T h e c o u n t s are related to d r y w e i g h t as well as to volatile d r y solid m a t t e r . T h e e r r o r b a r s listed refer to 1 cr e s t i m a t e s (n - 1).
Heterotrophic saprophytes Bacteria c o u n t s o f total h e t e r o t r o p h i c s a p r o p h y t e s in t h e biofilm o r i g i n a t i n g f r o m d o m e s t i c w a s t e w a t e r are in the r a n g e o f t h o s e f o u n d in a c t i v a t e d s l u d g e
Table 1. Population densities of various physiological groups of heterotrophic bacteria originating from both sewer biofilms and activated sludges, given as colony forming units (cfu) and most probable number (MPN) related to g dry weight (DW) and volatile dry solid matter (vDW), respectively. The error bars listed refer to 1 a estimates (n - 1) Biofilm from domestic wastewater (n=5-10) Heterotrophic bacteria Proteolytic bacteria
cfu cfu cfu cfu Polysaccharide cfu degrading bacteria cfu Lipolytic bacteria cfu Ammonifying bacteria Nitrate reducing bacteria Denitrifying bacteria
per per per per per per per
10I° 7.3 x 101°+3.3 x 10~° 10~° 1.3 × 10~+6.3 x 10~° l09 2.5 x 10t° + 5.6 × 10t° 109 3.2x 10~°+ 6.7 × 10~° 109 2.1 x 10~°+ 1.5 x 10~° 10t° 3.3 × 10~°+ 2.1 × 10~° 109 2.0 × 10t° _+2.8 x 10]° cfu per g vDW 5 . 3 x 1 0 9 5.1 x l09 2.5 × 10~°+3.3 × 10I° cfupergDW l.l×10t°__l.3×10 t° 1.4×10H+1.7×10 tl cfupergvDW 1.8×10~°+2.2×10 ~° 2 . 6 x l O H + 3 . 5 × I 0 H MPN per g DW 2.8 × 10tl +3.6 × 10H 8.5 × 1011+8.1 × 10H MPN per g vDW 4.4× 10H___6.4 × 10II 1.4x I012+1.3 × 10~2 MPN per g DW 3.0× 10]°_+2.7x 10r° 3.0× 10~-+3.6 × 10H M P N p e r g v D W 4 . 3 × 1 0 t ° + 3 . 0 x 1 0 ~° 4.3xlOH_+5.1×I0 ~t
*Single determination; n = 1.
g g g g g g g
DW vDW DW vDW DW vDW DW
1.3 × 10t°~ 1.3x 2.4 x 10~°_+2.4 x 3.7 x 109 + 5.0 × 5.5x 109_+6.6× 8.1 x 1 0 9 7.2 × 1.3 x 10~°___1.2 x 2.7 x 109 + 1.8 ×
Biofilm from trade wastewater (n =5 10)
High load activated sludge (n= 10-15) 2.7× 10t°+ 1.1 × 10t° 3.8 × 10t°_+ 1.6 x 10]° 1.6 × 109* 2.2x 109 8.1 × 10~°* I.I × 10II 1.4 × 10~°* 1.9x 10~° 1.3 ×10~° _+8.6 ×109 1.7 ×10~° -+1.2 ×10 t° 3.0 × 10~°_+2.2 × 10I° 4.0× 101°+2.9 × 10I° 3.1 × 109+4.8× 109 4.0 x l09 -+ 6.3 ×109
Low load activated sludge (n =10-15) 1.8 x 109± 1.7 × 109 3.3×109_+3.0×109
6.5 × l0 s* 1.2× 10~ 5.9 x 103* 1.1 × 10I° 6.5 x 10~* 1.2×109 5.1 ×10s_+3.1xl0 s 9.5×10s+6.3×10 s 2.6 × 109±2.5 x 109 4.7× 109--+4.6× 109 2.3× 10s-+2.3 x l0 s 4.3×10~+4.2×10 s
Sewer biofilms and activated sludge from the high load stage. The counts in the biofilm from trade wastewater are one order of magnitude higher. The lowest counts were found in the low load stage. Gutekunst (1989) reported a density of heterotrophic bacteria of 109 cfu g ~volatile dry solid matter in both sewer biofilms originating from domestic wastewater and biofilms from heavy metal contaminated wastewater. These counts are quite low compared to our results. In contrast to our results in the investigations of Gutekunst the count of heterotrophs did not differ between the domestic wastewater biofilm and the biofilm from industrial wastewater. However, the author found a 10-fold higher population density of aeromonads, pseudomonads and clostridia in the domestic sewer compared to the industrial one. In activated sludge the density of heterotrophic bacteria was determined by Prakasam and Dondero (1967) as 2.6x 108cfuml -~, i.e.--related to dry weight--in the same range as those found in the high load stage of the municipal plant investigated here. The counts reported by G/ide (1982) were in the same order of magnitude (108 cfu ml-~). Benedict and Carlson (1971) found densities of heterotrophic bacteria of 2 x 10~1-5 x 10Hcfug -t dry weight in the activated sludge of a municipal sewage plant. Schmider and Ottow (1986) found bacterial numbers between 2 x 106 and 2 x 107 MPN ml i for heterotrophic bacteria from the aeration tank of a low load municipal plant. Wetzel and Dott (1984) reported average counts of heterotrophic bacteria of 4.8 x 108 cfu mlin activated sludge from the high load stage (F:M ratio 4.3 kg kg i d- 1) and of 4.8 × l07 cfu ml-l in the low load stage (F:M ratio 0.13kgkg 1d-l) of a two-stage municipal plant treating wastewater with 40% domestic and 60% trade/industrial wastewater. The MLSS were determined as 2.8 and 3.5gl ~, respectively. Thus the counts translate to 1.7 x 10H and 1.4 x 101°cfu g 1 dry weight. The counts in the high load and in the low load sludge differ in one order of magnitude as did the counts in the present study. K~impfer (1988) found a density of heterotrophic bacteria of 1 x 107-9 x 107cfuml -~ in the activated sludge of a one-stage plant (F:M ratio 0.25 kg kg-~ d-~; MLSS 3.3gl ~), i.e. 3 x 109-2.7 × 10J°cfug ~ dry weight. These values agree with the values we found in the activated sludge of the two-stage municipal plant.
Polymer degrading bacteria: proteolytic bacteria, polysaccharide degrading bacteria and lipolytic bacteria The counts of polymer degrading bacteria were in the range of 109-101° in both biofilm biocenoses and the high load activated sludge. Those in the low load sludge were one order of magnitude lower, similar to those of heterotrophic bacteria. Literature data on the densities of proteolytic and lipolytic as well as on polysaccharid degrading bac-
1343
teria are scarce. Dott (1980) reported on heterotrophic bacteria populations in slime originating from drinking water systems. The counts for gelatinase-positive bacteria were in the range of 10-20% of the count of total heterotrophs. Dott and Wetzel (1984) found higher counts of "polymer degrading" bacteria in the sludge from the high load stage of a two-stage wastewater treatment plant compared to the low load sludge. This is in agreement with our results. The authors stress that the bacteria from the high load milieu are physiologically more flexible, many of them are able to degrade polymers. Gfide (1982) found that the total counts of "lytic bacteria" (i.e. lysis of polysaccharides such as cellulose, chitin, dextran, pectin and xylan and lysis of gelatine) in wastewater biocenoses are always at least one order of magnitude lower than those of heterotrophs assessed on casitone-yeast agar.
Bacteria of the nitrogen cycle: ammonifiers, nitrate reducers and denitrifiers This group developed high population densities in both the DB- and the TB-biocenosis. They were in the same range or even higher than in the high load activated sludge. In the low load stage average counts of ammonifiers were quite low. The counts of gas forming "true denitrifiers" in both activated sludge samples were about one order of magnitude lower than the counts of nitrate reducers. Compared to the densities found in the biofilm biocenoses the counts in the low load sludge are two to three orders of magnitude lower. The high oxygen concentration as well as the limitation of carbon sources in the low load stage do not support denitrification processes. In "classical denitrification" the enzyme system is induced by anoxic conditions only. In contrast, aerobic denitrification with a constitutive enzyme system was observed only in a few cases (Robertson and Kuenen, 1989). This could be a reason for the low density of the heterotrophic denitrifying population we found in the low load activated sludge community. Schmider and Ottow (1986) investigated the bacteria populations in activated sludge from a low load wastewater treatment plant. In the aeration tank they found between 2 x 106 and 2 x 107MPNml ~ of heterotrophic bacteria, the density of (gas producing) denitrifiers was in the range of 5 x 105 and 1.1 x 107 MPN ml -~. The MPN-assessment of denitrifying bacteria is methodologically problematic. The inoculum in the test tube is a mixed culture of assimilatory and dissimilatory nitrate reducers and "true denitrifiers" as well as aerobic denitrifiers, all competing for the nitrate. "True denitrifiers" may be occasionally outcompeted. This leads to "false negative" results with "improbable combinations of positive and negative results", if gas production is chosen as a test criterium.
H1LDE LEMMER et al.
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Table 2. Enzyme activities of various physiological groups of heterotrophic bacteria from sewer biofilms and activated sludges, given as % substrate turnover (a) and as mU (b) related to mg dry weight (DW) and mg volatile dry solid matter (vDW), respectively. The error bars listed refer to I cr estimates (n - 1)
(a)
Esterase cc-Glucosidase /~-Glucosidase Phosphatase L-Ala-aminopeptidase
% 0/° % 0/° % % % % % %
turnover turnover turnover turnover turnover turnover turnover turnover turnover turnover
per per per per per per per per per per
h h h h h h h h h h
and and and and and and and and and and
mg mg mg mg mg mg mg mg mg mg
DW vDW DW vDW DW vDW DW vDW DW vDW
Biofilm from domestic wastewater (n = 5-10)
Biofilm from trade wastewater (n ~ 5-10)
High load activated sludge (n = 5-10)
Low load activated sludge (n = 5-10)
42.3 + 21.0 69.3 4- 44.0 4.5 4- 2.5 7.0 4- 2.6 5.3 _+ 1.6 9.1 4-4.8 18.1 4- 8.8 29.3 4- 10.5 34.5 4- 24.2 53.4_+28.0
95.6 4- 50.8 136.7 _+ 79.4 9.4 + 4.8 12.4 4- 6.4 14.0 4- 11.0 18.24- 13.2 32.1 ___16.5 43.4 4- 25.5 93.1 4- 47.2 123.44-63.4
51.1 4- 14.6 67.8 4- 18.8 6.3 4- 1.5 8.4 4- 2.0 4.6 + 0.9 6.2+ 1.2 17.9 4- 3.2 24.0 4- 4.2 113.7 4- 34.6 152.04-46.7
21.0 4- 5.8 37.1 4- 8.4 1.6 4- 0.3 3.0 4- 0.5 1.5 4- 0.3 2.84-0.6 6.3 + 1.5 11.5 4- 2.8 39.1 4- 3.6 71.24-7.7
Biofilm from domestic wastewater (n = 5-10)
Biofilm from trade wastewater (n = 5-10)
High load activated sludge (n = 5-10)
Low load activated sludge (n = 5-10)
1.4 + 0.7 22 ± 1.4 2.5 4- 1.4 3.9 4- 1.4 2.9 +_0.9 5.04-2.7 8.1 4- 4.0 13.2 4- 4.7 23.4 4- 16.4 36.24- 1 9 . 0
3.1 4- 1.6 4.4 4- 2.5 5.2 + 2.7 6.9 + 3.6 7.7 _+ 6.1 I0.1 4-7.3 14.4 4- 7.4 19.5 4- 11.4 63.2 4- 32.0 83.74-43.0
1.6 4- 0.5 2.2 4- 0.6 3.5 4- 0.8 4.7 + 1. t 2.6 4- 0.5 3.44-0.7 8.1 4- 1.5 10.8 4- 1.9 77.1 4- 23.5 103.1 4-31.7
0.7 4- 0.2 1.2 4- 0.3 0.9 4- 0.2 1.7 4- 0.3 0.9 4- 0.2 1.64-0.3 2.8 + 0.7 5.2 4- 1.2 26.5 4- 2.4 48.34-5.3
(h)
Esterase • -Glucosidase /~-Glucosidase Phosphatase L-Ala-aminopeptidase
mU mU mU mU mU mU mU mU mU mU
per per per per per per per per per per
mg mg mg mg mg mg mg mg mg mg
DW vDW DW vDW DW vDW DW vDW DW vDW
Enzyme activity of various populations of heterotrophic bacteria Enzyme activities of various physiological groups of heterotrophic bacteria from both sewer biofilms and activated sludges are shown in Table 2. In Table 2(a) the activities are given as % substrate turnover h -1, in Table 2(b) as mU. The values are related to mg dry weight and mg volatile dry solid matter, respectively.
Esterase activity Esterases are unspecific enzymes involved in the degradation of polymers. Esterase activity was determined in order to assess the total heterotrophic degradation activity of sewer biofilms and activated sludges. In the TB-biocenosis about 2-fold higher activities were found compared to the DB-biocenosis. This is in contrast to results of Gutekunst (1989) who found significantly higher activities of dehydrogenase and oxygen consumption in domestic sewer biofilms compared to industrial ones. The esterase activity of the activated sludge biocenosis from the high load stage was in the same range as the activity of the biofilm in the domestic sewer. Low load activated sludge was only half as active as the forementioned biocenoses. Esterase activities in biofilm bioeenoses from the sediment of the river Neckar/FRG were determined by Altmeier and Schweisfurth (1989) as 1-5/~ U mgdry weight. The activities of wastewater biocenoses
studied in the present work were three orders of magnitude higher.
Activity of ~-glucosidase and t~-glucosidase Glucosidases hydrolyse disaccharids originating from the degradation of polysaccharids, cc-Glucosidase cleaves the disaccharides maltose and saccharose. //-Glucosidase as a part of the cellulolytic enzyme system cleaves cellobiose. The activity in the TB-biocenosis was 2-fold higher, the one in the high load activated sludge in the same range as in the DB-biocenosis. The low load activated sludge showed a low activity, about a third of the DB-activity. A similar activity pattern was determined for the/~-glucosidase. Teuber and Brodisch (1977) reported glucosidase activities in activated sludge from three one-stage wastewater treatment plants in the range of 5.2-16.0 mU mg -~ protein. Assuming a protein content of about 50% of the dried solid matter (N/iveke and Tepper, 1979), the glucosidase activities are between 2.6 and 8.0mU/mg -j dry weight, i.e. compared to the activities related to volatile dry solid matter, in the same range as those found in this study for the biofilm biocenoses. Verstraete et al. (1976) found the saccharase activity to be specifically related to the organic matter content, i.e. organic pollutants such as proteins and carbohydrates. Random samples taken from the wastewater revealed somewhat higher COD values in the trade wastewater than in the
1345
Sewer biofilms and activated sludge domestic one. High maximum values suggest shock load conditions in the trade sewer compared to the domestic sewer.
decreased to 20% after 4 days of starvation. Thus the low values found by Nybroe et al. (1992) might be attributed to starvation effects induced by the low load of the plants they investigated.
Phosphatase activity
Phosphatases are esterases cleaving phosphomonoesters. They have a low substrate specifity and describe the total metabolic activity of a biocenosis. The activities of phosphatase found in the biofilm and activated sludge biocenoses show a similar pattern to the glucosidases. The activities found in the DB-biocenosis and the high load activated sludge are in the same range, the TB-biocenosis had a 2-fold higher activity. The low load activated sludge biocenosis was only a third as active as the organisms of the high load stage. Phosphatase activity is known to be related to eutrophication. The higher the degree of eutrophy the higher phosphatase activities were found (Hantke and Melzer, 1991). Verstraete et al. (1976) found heavy loads of organic pollutants in general and proteinaceous compounds in particular to induce increased phosphatase activity. Activity of L-alanine-aminopeptidase
Protein metabolism plays an important role in microbial communities, especially in the degradation of decaying biomass. Peptides are cleaved by aminopeptidases. These exopeptidases are specific for different amino acids, e.g. for alanine. The activity pattern of L-alanine-aminopeptidase in the biofilm and activated sludge biocenoses differed from that determined for the glucosidases and phosphatases. The activity in the DB-biocenosis was in the same range as in the low load activated sludge biocenosis. Both the TB-biocenosis and the high load activated sludge, however, showed a 3-fold higher activity. Teuber and Brodisch (1977) reported peptidase activities in three activated sludges in the range of 28.2, 55.3-76.3 m U m g -1 protein. Assuming a protein content of 50% in the dry matter of biomass (N/iveke and Tepper, 1979), we get around 14.1, 27.7 and 38.2 mU m g -1 dry weight. Brodisch et al. (1979) found a peptidase activity of 5 mU mg ~ dry weight in the return sludge from a wastewater treatment plant with a F: M ratio of about 0.2 kg kg-~ d - l . This activity is quite low compared to the activities in the biocenoses studied here. Nybroe et al. (1992) found for low load activated sludge from plants in Denmark that the peptidases show the highest substrate production rates of the enzymes investigated here. But the activity ofesterase and the glucosidases they report are lower than the values we found. However, the characteristics of the plants Nybroe et al. investigated are somewhat different. Generally we found enzyme activities higher than the values reported in the literature. Teuber and Brodisch (1977) found that specific enzyme activities
CONCLUSIONS
The population density of heterotrophic saprophytes we found in high load and low load activated sludge is similar to that reported in various other publications. The high load in the first stage of a two-stage wastewater treatment plant yielded one order of magnitude higher counts of heterotrophic bacteria compared to the low load second stage. Biofilms from sewers discharging domestic and trade wastewater revealed one to two orders of magnitude higher counts for the heterotrophic bacteria compared to high load and low load activated sludges. Enzyme activities show similar patterns in the substrate turnover rates of esterase, ct- and fl-glucosidase and phosphatase in the biofilm biocenoses as in activated sludge. However, the turnover rate of L-alanine-aminopeptidase is very high in the high load activated sludge. Sewer biofilms are fixed film systems. Their ability to efficiently remove carbon and nitrogen from wastewater is well documented. For many years the most prevalent applications of these systems are carbon and nitrogen removal (Bryers and Characklis, 1990). We found population densities and enzyme activities which reveal the sewer biofilms to be a highly active biocenosis. Their heterotrophic activity is comparable with the one in high load activated sludge or even exceeds it. Compared to suspended microorganisms those attached to biofilms are known to be more resistant to toxic materials such as heavy metals. The high activity we found for the bacteria in the TB-biocenosis exposed to high concentrations of chromium and nickel confirms this statement. We find highly developed eukaryotic organisms such as slime molds and a variety of proto- and metazoa to be abundant in the DB-biocenosis. In the TB-biocenosis, on the other hand, they occur only in low numbers or else are even lacking. Eukaryotic organisms, in contrast to bacteria, seem to be much more susceptible to pollution by heavy metals. The combination of determining both population densities and enzyme activities separately for specialized groups of bacteria turned out a useful and workable tool to describe the activity of a biocenosis. For biology to efficiently support the development and performance of wastewater treatment, more data of that kind are needed for prokaryotic as well as eukaryotic organisms from various wastewater biocenoses such as activated sludge from stabilization tanks, trickling filter biofilms, fluidized bed systems or biomass support particle systems.
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HILDELI~MMERet al.
Acknowledgements--We wish to thank Dagmar Laschka and Christine G~inther from the BayLWF for the heavy metal analyses by ICP--OES. We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft (Biofilm Project, Grant Le 724/1-1).
REFERENCES
Altmeier U. and Schweisfurth R, (1989) Untersuchungen zur mikrobiellen Charakterisierung yon uferfiltriertem Grundwasser. Vom. Wass. 73, 333-444. Benedict R. G. and Carlson D. A. (1971) Aerobic heterotrophic bacteria in activated sludge. Wat. Res. 5, 1023-1030. Brodisch K., Teuber M. and Hegemann W. (1979) Enzymaktivit/iten als Kennwerte des belebten Schlammes. GWF 120, 524-527. Bryers J. D. and Characklis W. G. (1990) Biofilms in water and wastewater treatment. In Biofilms (Edited by Characklis W. G. and Marshall K. C.), pp. 671-696. Wiley, New York. Daubner I. and Ritter R. (1972) Zur Methodik des quantitativen Nachweises yon ammonifizierenden und denitrifizierenden Bakterien im Wasser mit Anwendungsbeispielen (Baggersee-Untersuchungen). Int. Rev. ges. Hydrobiol. 57, 517-522. Dott W. (1980) Qualitative und quantitative Bestimmung von Bakterienpopulationen aus aquatischen Biotopen: 1. Mitteilung: Selektionierung yon Bakteriengruppen durch Isolierung auf verschiedenen N~ihrb6den. Zbl. Bakt. LAbt. Orig. B 170, 93-98. Dott W. and Wetzel A. (1984) Mikrobiologische Untersuchungen einer zweistufigen Adsorptionsbelebungsanlage: 2. Mitteilung: ln-vitro-Aktivithten der isolierten Bakterien. Z. Wass.-Abwass.-Forsch. 17, 182-185. Giide (1982) Interactions between floc-forming and non floc-forming bacterial populations from activated sludge. Curr. MicrobioL 7, 347-350. Gutekunst B. (1989) Praktische Erfahrungen und Ergebnisse aus Sielhautuntersuchungen zur Ermittlung schwermetallhaltiger Einleitungen. Korr. Abwass. 36, 1367-1375. Gutekunst B. and Hahn H. H. (1985) Schwermetalle in Sielh/iuten--Eine Mfglichkeit zum Nachweis von Einleitungen schwermetallhaltigen Abwassers in die Kanalisation. Vom. Wass. 65, 127-137.
Hantke B. and Melzer A. (1991) Phosphataseaktivit/it in drei Seen unterschiedlichen Trophiegrades. Verh. Ges. OkoL (Freising-Weihenstephan 1990). 20, 513-520. K/impfer P. (1988) Automatisierte Charakterisierung mikrobieller Lebensgemeinschaften. Hyg. Berl. 1, 1-248. Laschka D. and Trumpp M. (1991 ) Sielhautuntersuchungen zur Lokalisierung von AOX-Emittenten im Kanalnetz. Korr. Abwass. 38, 495-496. Lemmer H. (1985) Mikrobiologische Untersuchungen zur Bildung yon Schwimmschlamm auf Kl/iranlagen. Ph.D. thesis, Technical University Munich, Germany, N/iveke R. and Teppcr K.-P. (1979) Einfiihrung in die Mikrobiologischen Arbeitsmethoden mit Praktikumsaufgaben. Fischer, Stuttgart. Nybroe O., Jorgensen P. E, and Henze M. (1992) Enzyme activities in wastewater and activated sludge. Wat. Res. 26, 579-584. Obst U. and Holzapfel-Pschorn A. (1988) Enzymatische Tests fiir die Wasseranalytik. Oldenbourg, Miinchen. Pitt T. L. and Dey D. (1970) A method for detection of gelatinase production by bacteria. J. appl. Bact. 33, 687-691. Prakasam T. B. S. and Dondero N. C. (1967) Aerobic heterotrophic bacterial populations of sewage and activated sludge. I. Enumeration. Appl. Microbiol. 15, 461-467. Reichardt W. (1978) Einfiihrung in die Methoden der Gewiissermikrobiologie. Fischer, Stuttgart. Rhodes M. E. (1959) The characterization of Pseudomonas fluorescens. J. gen. Microbiol. 21, 221-263. Robertson L. A. and Kuenen J. G. (1983) Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium. J. gen. Microbiol. 129, 2847-2855. Schmider F. and Ottow J. C. G. (1986) Charakterisierung der denitrifizierenden Mikroflora in den verschiedenen Reinigungsstufen einer biologischen Kl/iranlage. Arch. Hydrobiol. 106, 497-512. Tan T. L. and Overbeck J. (1973) Okologische Untersuchungen iiber nitratreduzierende Bakterien im Wasser des PluBsees (Schleswig-Holstein). Z. Allg. Mikrobiol. 13, 71-82. Teuber M. and Brodisch K, E. U. (1977) Enzymatic activities of activated sludge. Eur. J. appl. Microbiol. 4, 185-194. Verstraete W., Voets J. P. and van Lancker P. (1976) Evaluation of some enzymatic methods to measure the bio-activity of aquatic environments. Hydrobiologia 49, 257-266.