Microbiological characterization of some wastewater sludges

Agricultural Wastes 12 (1985) 137 145

Microbiological Characterization of Some Wastewater Sludges L. Allievi & A. Ferrari |stituto di Microbiologia Agraria e Tecnica, UniversitY,degli Studi di Milano, Milan, Italy

ABSTRACT The most economical and ecologically satisfactory method .]'or the disposal o[ wastewater sludge is agricultural land application. This requires, however, a preliminao' chemical and microbiological characterization g/ the sludge. In this work the number of microorganisms belonging to the main soil and fecal groups in ten sludges from various sources were determined. Known chemical features of the sludge are also reported. The C02 production in soil portions amended with.five of the most representative sludges was also evaluated. Microbiological analyses showed that not only the physiological group counts but also the fecal group ones were very high, even in industrial sludge. The danger represented by this latter presence should, therejbre, always be considered. The CO 2 production test showed that all sludges stimulated soil microbial activity; marked differences among sludges were, however, jbund.

INTRODUCTION Increasing preoccupation with pollution problems, added to the increase of waste accumulation, is responsible for the development of wastewater treatment plants. This development emphasized the problems concerning the disposal of sludge, an unavoidable by-product of these plants. The economically and ecologically most effective method of disposal is land application, possibly as fertilizer for cultivated land, generally requiring organic matter. However, it is clear that such a possibility of use requires 137 Agricultural Wastes 0141-4607/85/$03.30 c~ Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain

8.0 0. I 2.0 ND* 31.4 22.0 40.0 47.0 402.2

1.1

22.8 1.3

A

’ For origin of sludges corresponding * ND = not determined.

Dry matter (“4) Ash ( %) Total nitrogen (7;) Total P,O, (%) K,O (%) Cd (ppm) Co (ppm) Cr (ppm) Cu (ppm) Ni (ppm) Pb (ppm) Zn (ppm)

__~ 11.3 4.2 3.1 1.4 0.1 ( < 4.4) ND 53.0 4.4 74.2 38.3 1 369.0

C

Features

12.4 3.5 4.9 7.9 0.4 4.7 ND 380.0 2 100.0 75.5 205.0 7 650.0

D 7.8 0.9 8.6 10.9 0.8 1.7 ND 32.0 16.5 11.4 18.3 116.6

E F 8.5 36.2 3.4 7.2 0.1 2.4 10.0 33.0 32.3 31.0 71.7 754.2

Sludge”

25.8 15.7 3.2 1.7 0.0 I.0 ND 6.0 24.0 ND 3.8 122.0

G

TABLE 1 of the Sludges (values on dry matter)

to letters see Methods.

37.0 53.7 1.8 3.5 0.5 2.3 7.0 12.0 19.0 13.0 15.0 331.0

B

Chemical

13.8 3.9 5.2 1.6 0.2 53.6 ND 29, I 66,1 23.2 29.1 6061.0

H

17-l 2.6 5.2 2.4 0.4 2.0 ND 13.5 499.3 8.5 16.3 345.5

I

11.3 9.3 7.3 7.7 0.6 I.3 2.7 75.6 824.7 39.4 18.3 355.0

L

Microbiological characterization of wastewater sludges

139

as a preliminary an exact characterization of the sludge, not only from the chemical point of view (water, organic matter, heavy metal content, etc.) but also from the microbiological one. It is useful to obtain information on numbers of microorganisms belonging to both physiological and fecal groups; the latter are indicators of the possible presence and survival of truly pathogenic intestinal microorganisms. It is well known that fecal microorganisms, even if present in an effluent in low numbers, tend to concentrate, or even multiply, in wastewater sludge. The aim of our work was the microbiological characterization of ten sludges coming from both primary (sedimentation) and biological (activated sludge) plants treating civil, industrial and combined civil industrial effluents. The presence of aerobic and anaerobic heterotrophic bacteria, of nitrogen and carbon cycle microorganisms, and of the most important fecal bacteria was determined. The principal physical and chemical features of all the sludges were already known. To obtain information on effects of land application of the studied sludges, the in vitro CO 2 production (proportional to total biochemical activity) of soil portions amended with five of the samples, chosen as representative was evaluated. The additions corresponded to a 10 t/ha treatment. METHODS

Sludge samples The samples and their chemical analyses reported in Table 1 were supplied by the Istituto di Chimica Agraria of the University of Milan. The sludge sources were: Sample A : primary sedimentation p l a n t ~ e t e r g e n t industry B : primary sedimentation plant--meat canning industry C : activated sludge plant~--civil wastewater D: activated sludge plant---combined civil-industrial wastewater E : activated sludge plant--pharmaceutical industry F : activated sludge plant--pharmaceutical industry G : activated sludge plant--pharmaceutical industry (antibiotic production; effluent treated with CaO) H : activated sludge plant--chemical industry (amino-acid production for farm feed)

140

L. Allievi, A. Ferrari

I : activated sludge plant-~lye works L: activated sludge p l a n t ~ y e works

The samples were stored at 4°C in hermetically sealed plastic containers before analysis. Microbiological determinations

Ten grams (wet weight) of the sample were suspended in 90 ml of buffer solution (quarter-strength Ringer) and homogenized in an Omni Mixer (Sorvall). From this suspension, serial ten-fold dilutions in the same buffer were subsequently made. Duplicate and triplicate 1 ml volumes of appropriate dilutions were inoculated into agar media in Petri-dishes or in liquid culture media (count by M PN method), respectively. Anaerobic incubation was achieved by the Gas-Pak system (BBL). Dry weights of the sludges were determined by placing aliquots of the samples to dry to constant weight in an oven at 105 °C. The microbial groups determined the media and the incubation conditions were: (1) Aerobic heterotrophic bacteria: plate count agar (7 days at 28 °C). (2) Anaerobic heterotrophic bacteria: Todd Hewitt broth with 1.4 o~, agar (7 days in anaerobic conditions at 28 °C). (3) Ammonifiers: asparagine liquid medium according to Pochon & Tardieux (1962) (15 days at 28 °C); ammonium was detected using Nessler reagent. (4) Nitrifiers: ammonium liquid medium according to Pochon & Tardieux (1962) (20 days at 28°C): nitrite and/or nitrate were detected using diphenylamine H2SO 4 reagent. (5) Aerobic nitrogen-fixing bacteria (Azobacter): liquid medium according to Pochon & Tardieux (1962) (15 days at 33°C)~ the growth of A zobacter was confirmed by microscopic examination. (6) Anaerobic nitrogen-fixing bacteria: liquid medium according to Augier (1957) (30 days in anaerobic conditions at 28°C); the growth was revealed by rH lowering and abundant gas production. (7) Aerobic and anaerobic cellulolytic microorganisms: liquid media with strips of filter paper, according to Pochon & Tardieux (1962) (20 days at 28 °C for aerobic, and 33 °C in anaerobic conditions for anaerobic microorganisms).

Microbiological characterization of wastewater sludges

141

(8) Fecal coliforms: according to IRSA standard methods (1973, 1983); presumptive test: lactose broth (24 or 48h at 37°C); confirmatory test: brilliant green lactose bile broth, and tryptone water for indole production (24 h at 44 °C). Final confirmation was obtained by streaking Levine-EMB-agar plates. (9) Fecal streptococci: according to IRSA methods (1973, 1983); presumptive test: azide dextrose broth (24 or 48h at 37°C); confirmed test: ethyl violet azide broth (48 h at 37 °C). The growth of streptococci was confirmed by microscopic examination. (10) Sulfite-reducing clostridial (Clostridium perfringens group) spores: sulfite polymyxin sulfadiazine agar (Biolife) (48h in anaerobic conditions at 37°C); agar plates were inoculated with previously pasteurized (80°C/10min) dilutions; black colonies were counted. Soil respiration test (CO 2 production) The method of Pochon & Tardieux (1962) was used, modified as follows. Amounts of the tested sludge equivalent to 0.2 g dry weight were mixed with 60 g portions of sandy soil and placed in hermetically sealed glass jars. In order to absorb the CO2 produced, a beaker containing 40 ml of 0.5 Y N a O H was placed in each jar. Incubation was at 25 °C. Titration of the residual N a O H was with 0"5N HC1, after addition of 100ml of distilled water, 20 ml of 1 M BaCI 2 and six drops of phenolphthalein. CO 2 determinations were carried out after different incubation times; for each of these, three jars were prepared. As a control, unamended soil was tested.

RESULTS AND DISCUSSION The microbial counts obtained are reported in Table 2. Microbiological analyses show that all the sludges examined contained a considerable number of viable microorganisms. The numbers of heterotrophic bacteria and ammonifiers were very high and were similar among all samples, independent of the source of the sludge. On the contrary, microbial counts of most of the physiological groups were very different among the various sludges. With the exception of the aerobic nitrogenfixing bacteria, absent in most samples, nearly all of the main soil

A

B

C

" See Table 1. b nd = not detectable.

microorganisms Fecal coliforms Fecal streptococci Sulfite-reducing clostridial spores

microorganisms Anaerobic cellulolytic

1.9 x 104 7.3 x 103 7.3 x 105 1 . 5 x 105 1-8 x 106

32 nd 3-7 x 103 8.9 × 10 3 4.5 × 10 4 3.7 x 105 2 . 2 x 105 7 . 5 × 105 3 . 7 x 106

6"6 x 10 2 1"6 X 10 3 5.0 x 106

75

1.8 x 107

9-5 x 103

2"0 x 10 5

71 3-7 x 104 2 . 0 x 105

nd

2-2 x 101° 4.4 not

F x 10 9

G

5'0

× 10 2

nd 40 1.1 x 102

5.0 x 102

determined

9"1

X 10 3

nd 1.1 x 107 8 . 5 x 106

1.0 x 103

1.9 x 106

10 l° determined 2.7 x l0 s 109 9 . 5 x 107 1 . 5 x 109 103 9.6 x 102 6.4 x 102 105 nd nd not

2.1 x 7.7x 1-2 x 7.7 x

1.7 x 7.3x 3.5 x 7.0 x

109 108 107 102

5'6 x 101°

E

Sludge"

1.1 x 10 l°

D

4"2 x 10'*

32

Aerobic heterotrophic bacteria 2.5 x 109 8"0 × 10 9 3.4 x 108 Anaerobic heterotrophic 1"5 x 108 8'0 x 109 1"5 × 109 bacteria 3"4 x 109 4-5 x 109 5"0 x 107 Ammonifiers 2"5 x 103 nd 3.7 x 103 Nitrifiers nd nd 7.5 x 102 Aerobic N-fixing bacteria Anaerobic N-fixing 9"0 × 104 1"8 × 10 4 3-7 x 105 bacteria Aerobic cellulolytic

Microbial group

TABLE 2 Microbial C o u n t s o f the Sludges ( P F U or M PN/g dry weight)

× 1010

2"1

×

104

1.0 x 102 1.8 x 104 1 . 9 x 107

nd

4.1 x 105

4.8 x 101° 6 " 8 x 10 l° nd nd

8"2

H

× 10 9

2.2

x 10 4

50 3.2 x 106 4 . 8 x 104

1.8 x 102

5.4 x t05

1.0 x 109 1.2x 101° nd 38

8"5

1

5.6

x l0 3

50 3.7 x 100 7 - 9 x 103

1-2 x 103

2-2 x 105

3"3 x 10 l° 3"7 × 10 TM 3"7 × 105 nd

1-7 x 1011

L

Microbiological characterization of wastewater sludges

143

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l

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L

5 incubation time (days)

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I

I

I

10

Fig. I. C O 2 p r o d u c t i o n o f soil amended w i t h some sludges (each value is the mean o f three determinations). For sludges corresponding to lettering see Methods.

physiological groups were contained in each sample; the counts were comparable with, and often higher than, those generally found for soil. The cellulolytic microorganism groups represent the only case demonstrating a connection between the sludge source and the microbial count; the numbers of cellulolytic microorganisms in the civil and combined civil-industrial wastewater sludge were higher than in the others. The numbers of all these bacteria would be valuable in the case of agricultural land application. In the various samples the fecal bacteria were nearly always present in high numbers from a health standards point of view. This constant presence of fecal bacteria is worrying; the numbers were frequently very high even in sludge from industry. All this is consistent with the view that the pathogenic microorganisms problem is one of the most serious in land disposal of sludge. Respiration test results are shown in Fig. 1. All the tested sludges induced increases in soil metabolic activity, with marked differences among the samples. In the case of samples F and L the increases in CO 2 production are small, while the CO2 production induced by sludges B, C and G is about twice that in the control sample (unamended soil). Sludge B gave the greatest stimulation, although it was the poorest in organic matter. In the sludges studied, the heavy metal concentration generally did not exceed the limits fixed by some European countries for land disposal; in fact, only the amount of Cu present in sample L exceeds some

144

~L. Allievi, A. Ferrari

TABLE 3 Some Limits Fixed in European Countries for Metals in Sludge (mg/kg dry weight) (Commissione delle Comunitfi Europee, 1982)

Metal

Cd Co Cr Cu Ni Pb Zn

West France Netherlands German)' 30 nff 1200 1200 200 1 200 3000

15 20 200 1500 100 300 3000

10 nf 500 600 100 500 2000

Denmark

30 nf 500 700 nf 1200 6000

Finland Norway Sweden Belgium

30 100 1000 3000 500 1200 5000

15 50 nf nf nf 300 nf

15 50 1000 3000 500 300 10000

10 20 500 500 100 300 2000

a nf = not fixed.

of these limits (Table 3). On the other hand, with the adopted experimental conditions it may be calculated that the soil Cu concentration increase consequent upon sludge L addition is only about 3 mg/kg. The Cu concentration normally present in soil is said to be 5-20 mg/kg, and the limit 100 mg/kg (Table 4). With regard to the other metals and sludge, the situation is evidently even less alarming. So it may be supposed that in our test heavy metals cannot have affected microbial activity and therefore the production of CO 2. The increase in CO 2 production observed after adding the samples to the soil might also be ascribed to scanty nutritional characteristics of the soil used. The TABLE 4 Heavy Metal Content of Soils (mg/kg dry weight) (Commissione della Comunit~i Europee, 1982)

Metal

Cd Co Cr Cu Ni Pb Zn

Mean concentration normally .[ound in soil 0-1 I 10 5 10 0.1 I0

I 10 -50 20 50 20 50

° Proposed in West Germany.

Limit"

3 100 100 50 100 300

Microbiological characterization of wastewater sludges

145

noticeable disparities in stimulation among the sludges might be mainly attributed to the different biodegradabilities of organic matter of the various samples. It is reasonable to believe, for example, that the high CO 2 production in soil amended with sample B, consisting of matter not yet treated in a biological plant, is due to the high biodegradability of the organic matter contained in this sludge. In sludges such as we have studied, the high number of fecal microorganisms seems to be the most important problem. The laws of the various countries, even if taking into consideration this problem, generally do not prescribe numerical limits, except for the Swiss law which prescribes a maximum of 100 enteric bacilli/g sludge for land disposal (Consiglio Federale Svizzero, 1981). It would be opportune for legislators to take more care of this aspect of sludge disposal.

REFERENCES Augier, J. (1957). Apropos de la fixation biologique de l'azote atmospherique et de la numeration des Clostridium fixateurs dans les sols. Annales de l'Institut Pasteur, 92, 817-24. Commissione delle Comunit~ Europee (1982). Proposta di Direttiva del Consiglio concernente l'utilizzazione in agricoltura dei fanghi residuati dai processi di depurazione. COM(82) 527 def. Bruxelles, l0 Settembre. Consiglio Federale Svizzero (1981). Ordinanza concernente i fanghi di depurazione. 8 Aprile. Istituto di Ricerca Sulle Acque (CNR) (1973). Metodi analitici per le acque. Quaderni LR.S.A., 11(3), 37-55. Istituto di Ricerca Sulle Acque (CNR) (1983). Microorganismi indicatori di inquinamento fecale. In: Metodi analitici per i fanghi. Quaderni I.R.S.A., 64(1), 1-4. Pochon, J. & Tardieux, P. (1962). Techniques d'analyse en microbiologie du sol, Editions de la Tourelle, St. Mand6 (Seine).