Bioleaching of metals from sewage sludge: Microorganisms and growth kinetics

Wat. Res. Vol. 27, No. 1, pp. 101-110, 1993 Printed in Great Britain. All rights reserved

0043-1354/93 $5.00 + 0.00 Copyright ~) 1993 Pergamon Press Ltd

BIOLEACHING OF METALS FROM SEWAGE SLUDGE: MICROORGANISMS AND GROWTH KINETICS J. F. B L ~ , R. D. TYAGI*~ and J. C. AUCLAIR Universit6 du Qu6bec, Institut National de la Recherche Scientifique 0NRS-Eau), 2700 Rue Einstein, Ste-Foy, Qu6bec, Canada GIV 4C7

(First received November 1991; accepted in revised form May 1992) Abstract--Microbial leaching is one of the advantageous methods of removing heavy metals from sewage sludge, however, the microbiological aspects of this technology have not been studied. This study presents the characterization of the haturally occurring microorganisms, responsible for the metal leaching activity, in 21 different sewage sludges. The results obtained indicate that the bioleaching of metals is carried out by successive growth of less-acidophilic and acidophilie thiobaciUi. Several species of less-acidophilic thiohacilli participate in the sludge acidification, but Thiobacillus thioparus is the most important species. In contrast, Thiobacillus thiooxidans seems to be the only species involved in the acidophih'c group of thiohacilli. The growth kinetics of the two groups of thiobaciili was followed in five different sewage sludges. After 5 days of incubation in shake flasks, the pH of the sludge was decreased to about 2.0 and this pH decrease solubilized the toxic metals (Cd: 83-90%; Cr: 19-41%; Cu: 69-92%; Mn: 88-99%; Ni: 77-88%; Pb: 10-54%; Zn: 88-97%). The maximum specific growth rate (Pm~) for the less-acidophilic thiohacilli varied between 0.079 and 0.104 h -m and that for the acidophilic thiobaciUi varied between 0.067 and 0.079 h- 1.

Key words--metals, bioleaching, elemental sulfur, sewage sludge, thiobacilli, metal leaching, sulfur oxidation

INTRODUCTION

The increase in energy cost and renewed interests in sustaining a safe environment has resulted in increased attempts to utilize sewage sludge as fertilizer on agricultural land. In the U.S.A. and Europe, 40% of the sludge has been disposed by this method (Bruce and Davis, 1989; EPA, 1990). The presence of high concentrations of toxic metals in sewage sludge represents a major constraint in the utilization of sewage sludge as fertilizer. During wastewater treatment, the heavy metals are concentrated in the primary and secondary sludges (Stephenson and I.ester, 1987a, b). A substantial fraction of the influent metals is removed during primary treatment by precipitation (Sterrit and Lester, 1984). In activated sludge process (the secondary treatment), the metals are complexed with extracellular polymers and rendered insoluble (Brown and Lester, 1979). The metals are also absorbed by bacterial cells or physicaUy entrapped in the bioflocs (Stephenson and Lester, 1987a, b) during secondary treatment. These physical, chemical and biochemical mechanisms are responsible for the removal of 80-90% of the infinent metals in a wastewater treatment plant. The concentration of heavy metals observed in the sludge varies according to the type of wastewater treatment, the presence of certain metal processing industries in the concerned area,

population life style, runoff contamination and the state of the pipelines. In Canada and the U.S.A. more than 50% of the sludges produced exceed the obligatory recommended limits for toxic metals concentration for sludge agriculture use (St-Yves and Beaulieu, 1988; Wong and Henry, 1984; Wozniak and Huang, 1982). In the U.K. 82-85% of sludges do not meet the established guidelines (Lester et al., 1983). Therefore, the interest to extract sludge metals is very well established. The toxic metals in sewage sludge can be reduced by two methods: source control and metal removal from sludge. The source control is difficult to apply due to the difficulty in finding sources. This is also an expensive method. Moreover, even if it is possible to control all industrial metal discharges, the problem remains regarding the metals in domestic wastewaters (Davis and Jacknow, 1975; F6rstner and Wittmann, 1979; Klein et al., 1974; Tjell, 1986). For the last 20 years, several chemical methods have been examined to remove heavy metals from municipal sludge, but none of these methods are economical or efficient enough to remove all the concerned metals. This created an interest in the investigation of microbiological methods to remove heavy metals. Bioleaching of metals using bacterial strains of Thiobacillus ferrooxidans has been studied (Couillard and Mercier, 1991; Tyagi and Coulllard, 1989; Wong and Henry,

1988).

*Author to whom all correspondence should be addressed. 101

J . F . BLAISet al.

102

Recently, a simple microbiological process to remove heavy metals from sewage sludge has been examined. This process exploits the presence o f naturally occurring bacterial species in the sludge which are capable of oxidizing elemental sulfur into sulfuric acid (Blais et al., 1992a, b, 1993). This process is very efficient for the solubilization o f sludge toxic metals. However, the industrial exploitation o f this method requires knowledge of the nature o f microorganisms involved, the interaction between S-oxidizing and other bacterial populations present in the sludge, the kinetics o f bacterial growth and metabolic activity o f microorganisms. These aspects have been addressed in this paper. MATERIALS AND METHODS

Sewage sludge sampling The 33 sludges were obtained from 11 wastewater treatment plants in the provinces of Quebec and Ontario (Canada), and in the states of Delaware and Maryland (U.S.A.): St-Georges-de-Beauce (secondary sludge from an activated sludge unit); Beauceville, Black Lake, Cecil County, Cowansville, Granby and Ste-Claire (secondary sludge from an activated sludge unit and aeorbically digested sludge); Valcartier and Wilmington (secondary sludge from an activated sludge unit and anaerobically digested sludge); Toronto (primary sludge and primary and secondary anaerobically digested sludge); and St-Etiennede-Beaumont (oxidation pond sludge). Samples were collected in polypropylene bottles and kept at 4°C before

utilization. The sludge samples used in the experiments are presented in Table I.

Acclimation of sulfur-oxidizing microflora The accfimation of the sulfur-oxidizing microflora was carried out according to the technique of Blnis et al. (1993). 150 ml of each sludge sample were transferred to 500 ml capacity Erlenmeyer flasks. 1% (w/v) of tyndalized powdered elemental sulfur was added to all the flasks. All experimental flasks were incubated in a gyratory shaking incubator ('Model 26, New Brunswick Scientific Co.) at 28°C and 200 rpm for a period of 12-22 days for initial acidification (pH < 2.0). After the initial acidification, 150 ml of each fresh sludge combined with 1% tyndalized powdered elemental sulfur was mixed with 5% of the corresponding acidified sludge and then reincubated in the 8yratory shaking incubator. This step was repeated successively until the rate of pH reduction (initial pH 6-8) to 2.0 was maximiT~d over two consecutive transfers. At this stage, the indigenous sulfur-oxidizing microflora was assumed to be adapted. Trace metal bialeaching assays To test the trace metal leaching capacity of the adapted microflora (in their corresponding sludge), 200 ml of the five fresh samples (A, D2, L2, N, $3) were transferred to 500 ml capacity Erlenmeyer flasks combined with 0.5% powdered elemental sulfur. Each of the flasks was inoculated with 5% of their corresponding adapted sludge. The flasks were incubated in a gyratory incubator shaker at 28°C and 200rpm. 15ml samples were drawn at 12-h intervals for chemical analysis. The samples were centrifuged at 20,000 g for 15 min to separate sofids and liquids and the liquid portion (supernatant) was conserved to determine the solubilized trace metal and sulfate concentrations by plasma

Table 1. Description of the sludges used in this study Date Total solids Metal exceeding Code Origin Types (month year) (g/I) recommended levels* A Beauceville SEC 03/90 14.0 B Beauceville AER D 03/90 3.9 C Black Lake SEC 05/90 4.8 Cu C2 Black Lake SEC 05/91 3.2 Cu, Ni C3 Black Lake SEC 06/91 2.5 Cu, Ni (24 Black Lake SEC 07/91 3.2 Cu, Ni D Black Lake AER D 05/90 24.4 Cr D2 Black Lake AER D i 1/90 22.8 Cu D3 Black Lake AERD 04/91 21.9 Cu, Ni D4 Black Lake AER D 05/91 22.5 Cu D5 Black Lake AER D 06/91 20.4 Cu D6 Black Lake AER D 07/91 17.1 Cu E C,ecil County SEC 04/91 4.9 F Cecil County AER D 04/91 10.2 G Cowansviile SEC 03/90 14.8 Mn H Cowunsville AER D 03/90 30.4 Mn I Granby SEC 03/90 14.3 Cu, Mn J Granby AER D 03/90 31.3 Cu, Mn K Sainte-Claire SEC 06/90 1.7 Cu L Sainte-Claire AER D 06/90 40.7 L2 Sainte-Claire AER D 10/90 31.4 Mn M Saint-P.tienne OXID P 05/89 5.9 N Saint-Georges SEC 03/90 7. ! O Totonto PR ANA D 05/89 24.2 Cd P Toronto SEC ANA D 05/89 27.5 Cd Q Toronto PRIM 05/89 26.0 Cd R Valcartier SEC 08/90 2.8 Cu S Valcartier ANA D 09/89 14.0 Cu $2 Valcartier ANA D 06/90 15.6 Cu, Pb $3 Valcartier ANA D 08/90 13.2 Cu $4 Vaicartier ANA D 04/91 15.3 Cu T Wilmington SEC 04/9! 19.I Ni U Wilmington ANA D 04/91 28.2 Ni *Flynn et al. (1987). SEC - secondary activated sludge; AER D - acxobicaHydigestedsludp; OXID P =-sludgefrom oxidation pond; PR ANA D = primary sludge digested anaerobically; SEC ANA D - aecondary sludge digested anaerobically; PRIM = primary sludge; ANA D - anaerobically digested sludge.

Metal bioleaching from sewage sludge emission spectroscopy (ICP, Model Atom Scan 25 of Thermo Jarell Ash Corporation). To determine metal concentration in sludge, the latter was first digested in HNO 3, HF, and HCIO4 as per Standard Methods (APHA et al., 1989). Metal concentrations in sludge as well as recommended levels by the Qurbec Ministry of the Environment (Flynn et al., 1987) for agricultural use are presented in Table 7. Bacterial sampling The measurements of the thiobacilli in the sludges were carried out by a plate count technique using the S20~3synthetic salts agar medium ($M) for less-acidophilic (pH 7.0) and ac/dophilic (pH 4.0) thiobacilli described by Laishley et al. (1988) and Reynolds et al. (1981). The plate counts were carried out according to the modified technique of Dudley et al. (1980). Samples were obtained after vortex mixing I ml of sludge at high speed for 2 vain with 14 ml of sterile phosphate-buffered saline (0.01 M, pH 7.2) containing approx. 1 g of sterile 4-5 nun diameter glass beads in a 50 ml centrifuge tube. Sampleswere diluted serially in sterile phosphate-buffered saline, and 0.1 ml samples were spread with sterile glass L-rods over each of three replicate plates. Thiobacilli plates were incubated at 28°C for 2 weeks. The characterization of the leaching populations was carried out from the plate counts of fresh sludge samples (less-acidophillc thiobacilli) and sludges containing adapted microllora (acidophilic microtiora). A sample from each colony type was then purified by streaking and tested for pH lowering capacity in 0.5% $20]- or 0.5% tyndalized S° synthetic salts liquid medium (SM + $20]- or SM + S°) (Barton and Shively, 1968), during a 10 day incubation period. The ability of the acidophilic isolates to utilize a carbon source for heterotrophic growth was tested by supplementing the synthetic liquid medium (pH 4.0) with filter-sterilized glucose (1% final concentration), and the oxidation of ferrous ion as an alternate energy source was examined in 9K synthetic liquid medium (pH 2.5) containing 44 g/l of FeSO4 • 7H20 (Silverman and Lundgren, 1959). Bacterial growth in sludges and in synthetic medium A 5% inoculum of a 5-day old sub-cuiture (in SM + S°) of each standard American type culture collection strain (T. denitrificans ATCC 23644, T. neapolitanus ATCC 23640, T. intermedias ATCC 15466, 2", thiooxidans ATCC 19277 and ATCC 55128), less-acidophillc isolates (LA-D1, LA-LI, LA-NI) and acidophillc isolates (A-D1, A-LI, A-NI) were transferred to 150 ml of autoclaved sludges (D2 and $3) and synthetic medium (SM) amended with 0.5% tyndalized S°. The sludge pH was initially adjusted (with 2 N H2SO4 and 2 N NaOH) to pH 4.0 for acidophilic strains and to 7.0 for the less-acidophilic strains. The flasks were incubated at 28°C and 200 rpm during a 10 day period. RESULTS

103

in population of three different sludges sampled at different dates. The average concentration of thiobacilli in the 21 sludges sampled, the distribution of the two groups of thiobacilli according to the sludge solids content and the type of sludge (nondigested, aerobically digested, anaerobically digested) are presented in Table 3. There was no definite relation between thiobacilli content and these two parameters. It is necessary to note that the counts of two groups of thiobacilli are slightly inferior in anaerobically digested sludge than the two other types of sludge. Thiobacilli are strict aerobes (except T. denitrificans), the anaerobic digestion step will therefore tend to eliminate these microorganisms. After acclimation of the sulfur-oxidizing microflora, the population of acidophilic thiobacilli was increased to 10~-10s cfu/ml (Fig. 2). The acidification of all the sludges (pH < 2) diminished the lessacidophilic thiobacilli below the detection limit (Fig. 3). The concentration of acidophilic thiobacilli in acclimated sludge is not influenced by the sludge solids content and the type of sludge (Table 3). Characterization o f the leaching microorganisms

The characterization of sulfur oxidizing microflora has been carried out using the colonies of lessacidophilic thiobacilli in the sludge obtained on agar plates and acidophilic thiobacilli from the acclimated microflora in the sludge. A total of 98 colonies were isolated for the less-acidophilic group (3-6 different colonies per sludge). Ten colonies of this group did

(A) ~°6 i 10s

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ABCOEFGH

105":

'J K L'M N'O'P'Q'R'8'T'U

(8)

Enumeration o f sludge thiobacilli

The quantity of thiobaciUi present in the sludges has been evaluated. The population of lessacidophilic species in the sludge is uniform with a maximum variation of 1.7 log cycle [Fig. l(a)]. The measurement of acidophilic thiohacilli in the same sludges showed that these species were available in less quantity [Fig. l(b)]. For the majority of the sludges, the concentration of acidophilic thiohacilfi are below 10~ colony forming units (cfu)/ml. The variation of concentration of thiobacilli in the different type of sludges sampled at different times is shown in Table 2. There was no significant variation

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Sludges Fig. i. Distribution of the two thiobe.ciLll populations in sewage sludges. (A) Less-acidophilic and ~B) ac/dophllic.

104

J. F. BL~S et al. Table 2. Temporal variation of thiobacilli populatiom in sewage sludges Less-acidophilic (cfu/ml) x I04 Sludges Black Lake (secondary sludge)

Code

Concentration

C C2 C3

2.80 1.22 1.05 !.05

CA

Black Lake (aerobically digested sludge)

D D2 133 IM D5

D6 Valcartier (anaerobically digested sludge)

S $2 $3 $4

Mean 5: SD

Concentration

Mean + SD

1,53 + 0.85

1.48 !.50

1.25 + 0.28

9.18 + 5.60

4.40 3.80 3.65

2.73 + 1,47

19,2 6.80 4.73

Acidophilic (cfu/ml) x I0~

9.00 2.55

4.05

11.3 1.15 5.24 6.40 3.00

3.95 + 2.34 u

*Under detection limit (< 1.0 × I0J cfu/ml).

not grow on the agar medium. For the acidophilic thiobacilli, 26 colonies were purified from 21 different sludges. All the strains obtained were non-sporulated Gram-negative rods and did not contain visible cytoplasmic inclusions of sulfur. None of the acidophilic isolates has a capacity to oxidize iron to derive energy for growth and were incapable of growing in agar medium for heterotrophs. Based on the above-mentioned characteristics, the isolates can be classified as thiobacilli. The different species of Thiobacillus identified until today are presented in Table 4. The isolated species were inoculated in the SM + S° and SM + $2032- to verify the fimit of acidification in these media. This characteristic is an important criteria in the identification of thiobacilli (Hutchinson et al., 1969; Kelly and Harrison, 1988). All the acidophilic isolates decreased the pH of S M + S ° medium to 1.4-1.6 and SM + $20~3- medium to 1.9-2.2 in 10 days. For the less-acidophilic species, the limit of acidification was more variable between pH 2.2 and 6.9. Therefore, these isolates are distributed according to the groups established by Hutchinson et al. (1969) (Table 5). More than 63% of the isolates (56 out of 88) have been classified in the group which acidifies to pH 3.5--4.5. The only species of less-acidophilic thiobacilli which decreases the pH to 3.5--4.5 is T. thioparus (Kelly and Harrison, 1988). The other

isolates were distributed in five other groups. According to the taxonomic distinction proposed by Hutchinson et al. (1969) and Kelly and Harrison (1988) the composition of other groups of microorganisms is defined as follows: group 6 (pH 2.0-2.8) ffi T. intermedius; group 5 (pH 2.8-3.5)= T. neapolitanus, T. perometabolia, T. delicatus; group 3 (pH 4.5-5.5)ffi T. denitr~cana, T. novellus, T. tepi. darius; group 2 (pH 5.5-6.8)ffi T. versutus. The species of group 4 (final pH 3.5-4.5) were isolated from all tested sludges, the frequency of isolation was about 30% for the other groups. The percent distribution of the number of colonies of each group presented in Table 5 indicates an average proportion for the 21 sludges.. The thiobacilli of group 4 constitute more than 83% of the lessacidophilic microflora present in the 21 sludges sampied. The capacity of a few isolates to acidify two types of sterilized sludges (anaerobically and aerobically digested) was verified (Table 6). The acidification of sludges by pure ATCC cultures was also verified. The results obtained in the control sludge and the test sludges indicate that the indigenous species and those obtained from ATCC can grow in the sludge. Moreover, the acidification of sludges observed for all the species is comparable with the results obtained in the synthetic medium.

Table 3. Distribution of thiob~illi populations in the sludge groups

Acidopb~ic Sludges groups

Total solids (g/l)

Less-acidophilie (cfu/ml) × 104

Acidophilic (cfu/ml) x I03

(adapted microflors) (cfu/ml) x 107

Total (21)*

16.7 + I 1.3t

12.9 + 12.8

3.4 + 6.5

2.9 + 2.0

< 10 g/l (7) 10-20 g/l (6)

14,9 + 11.6

5.1 + 10.1

2.5 + 1.8

>20 g/! (8)

4.4 + 1.8 14.4-1-2.8 29.1 + 5.3

17.5+ 18.1 7.6 + 7.9

1.6+ 1.2 3.3 + 5.3

2.7+ 1.9 3.4 + 2.5

N.-dig. (11) Aer. dill. (6) Aria. dill. (4)

10.5 + 7.7 23.5 + 13.9 23.5 + 6.6

15.8 + 15.6 14.4 + 7.1 2.3 -I- 1.5

3.6 + 8.1 4.6 + 5.8 1.0 + 0.0

2.3 + 1.5 3.8 + 2.6 3.3 + 2.4

'Number of sludges used. ~'Mean + SD.

Metal bioleaching from sewage sludge

105

Table 4. List of species of the genus Thiobac//lus Thiobacilli groups

,°'1

Species

Reference

aguaesu//s

Wood and Kelly (1988) capsulatus Lidsldey et 01. (1988) delicatus Mizoguclu et 01. (1976) denitrO~cans Beijerinck (1904) intermediua London (1963) neapolitanua Parker and Prisk (1953) novellua Starkey (1934) perometabolis London and Rittenberg 0967)

1111°I!111111111111 Less-acidophilic

106

, , ,

, , i i , I i , , , , , i , ,

'AEOD'E'FGH

I J KLMNOPQRSTU

I

Sludges Fig. 2. Distribution of the acidophili¢ thiobacilli in sulfuradapted microflora from sewage sludges.

rubellus tep~larhts thermophilica thioparus thyasiris versutua

Mizoguclfi et oi. (1976) Wood and Kelly (1985) Williamsand Hoar¢ (1972) Beijerinch(1904)

acidophilus 01bertis

Guay and Silver (1975) Bryant et aL 0983) Huber and Stetter (1990) Temple and Colmer (1951) Reynolds et 01, (1981) Markosyan (1973) Huber and Stetter (1989) Waksman and Joffe (1922)

cupr/nus

Microbial growth and metal solubilization

The growth kinetics of thiobacilli and the toxic metal solubilization during leaching was followed in five different sludges (2 secondary, 2 aerobically digested and 1 anaerobically digested). For comparison the different types of sludges with varying solids content (7, 13, 14, 23, 41 g/i) were chosen. The composition of metals in the five sludges and those recommended by the guidelines (Flynn et al., 1987) is presented in Table 7. The metals which exceed the recommended limit in 33 sludges used in this study are listed in Table 1. More than 78% of the sludges (26 out of 33 sludges) have metals concentration incompatible with the norms for sludge use as fertilizer. Copper exceeds the limit most frequently (52%, 17 out of 33 sludges). A total of 7 o u t of 11 wastewater treatment plants sampled produced sludges which were higher than those recommended by the guidelines (Flynn et al., 1987).

ferrooxidags

Acidophilic

kabob/s

organoparus prosperus thiooxtdana

Wood and Kelly (1989) Harrison (1983)

In a 5 day period of microbial leaching, the sulfur oxidizing microfiora produced 2.8-4.4 g/1 of sulfate and this corresponds to 19-29% oxidation of added sulfur to sulfates (Table 8). The production of sulfuric acid decreased the sludge pH between 1.5 and 2.2. The decrease in pH and aeration of sludge resulted in an increase of the sludge redox potential (ORP). The metal solubilization after 5 days of leaching is presented in Table 9. The solubilization of cadmium, copper, manganese, nickel and zinc in 5 days was 86, 84, 94, 84 and 92%, respectively. However, chromium and lead were solubilized up to 34 and 32%, respectively. The growth curves of the two types of thiobacilli in digested sludge (Black Lake--sludge D2) are presented in Fig. 3. The less-acidophilic species initially 1°' ipresent in the sludge grows and decreases the pH to 4.0. At this pH, the acidophilic thiobacilli start multiplying and this results in appreciable sludge acidification. The decrease of pH below 3.0 causes a reduction in the less-acidophilic species population below the detection limit. The same phenomenon for growth and pH reduction was observed for four other sludges studied (not shown). For the five different sludges tested, the less-acidophilic species possess rapid growth with a generation time between 6.7 and 8 -8000 8.8 h (or p - 0.079-0.104 h-') (Table 10). The specific growth rate for acidophilic populations is lower with $ 4oo0 values between 0.067 and 0.079 h-'. The type of Zo4 3000 sludge and the solids content do not affect the growth a kinetics appreciably because the generation time (td) 2 2000 for the five sludges is comparable. At the same time, 1 -- 1000 the kinetics of sulfate production for five sludges is I I I I I I I I I 0 also comparable with a maximum sulfate production 0 12 24 $0 48 60 72 84 9 6 1 0 8 1 2 0 rate between 0.055 and 0.075g SO~- l -~ h -I and Time (11) maximum specific rate of sulfate production (Vm~) Fig. 3. Variations of the sulfur-oxidizingpopulations, pH and sulfate during metal leaching with Black Lake digested between 1.4 and 2.3 g S0~4- g-' cells h-'. The evalusludge (I)2). Symbols: O, pH; @, sulfate; A, acidophilic ation of the specific rate of sulfate production was thiobacilll; &, leu-acidophih'c thlobacilli. based on the assumption that I mg of dry weight

'°'k . . . .

\

106

J. F. BLAIS et al. Table 5. Proportions of the iess-acidophific groups of thiobacilfi in sewage sludges Less-acidophilic thiobacilli

pH final (SM + S°) and (SM + $20]-) 6.8-7.0 5.5--6.8 4.5-5.5 3.5-4.5 2.8-3.5 2.0-2.8

Group l Group 2 Group 3 Group 4 Group 5 Group 6

Isolate numbers

Isolation frequency (%)

Percent distribution mean -t-SD

6 6 7 56 7 6

28.6 28.6 33.3 100 33,3 28.6

2.2 _+4.3 2.6 + 4.9 6.2 :l: 10.1 83.4+_ 11.7 3.9 + 7.0 1.7 ± 3.1

Table 6. Acidification of autoclaved sludges and synthetic medium by pure cultures of thiobacilli after 10 days of incubation with 0.5°/@ of sulfur Final pH Strains

Synthetic medium

Autoclaved sludge D2

Autoclaved sludge $3

Control pH 7 (non-inocul.) denitrUic~ ATCC 23644 neapolitanus ATCC 23640 intermedius ATCC 15466 thioparm ATCC 8185 thioparus ATCC 55127 Isolate LA-DI Isolate LA-L1 Isolate LA-NI

6.85 5.20 2.90 2.08 3.80 3.74 3,78 3.82 3.69

7.04 4.80 2.80 2.30 4.02 3.94 3.86 4.18 3.85

6.94 5.30 3.12 2.62 4.05 4.36 4.29 4.40 4.25

Control pH 4 (non-inocul.) thiooxtdans ATCC 19377 thiooxidans ATCC 55128 Isolate A-D1 Isolate A-LI Isolate A-NI

3.97 1.54 1.48 1.52 1.48 1.59

3.96 1.50 1.46 1.66 !.66 1.62

4.04 1.77 1.70 1.72 1.72 !.68

oxidize thiosulfate slowly but do not possess the enzymatic system required for the oxidation of elemental sulfur: Aquaspidllum autotrophicum, Paracoccus denitrificans, Psuedomonas spp, Xanthobacter DISCUSSION autotrophicus, Achromobacter stutuzeri (Friedrich A number of bacterial species can derive energy for and Mitrenga, 1981; Tuttle et al., 1974). Certain their growth from the oxidation of elemental sulfur or recently isolated bacteria from soil (Arthrobacter spp, sulfur compounds. Several bacterial species found in Bacillus spp, Flavobacterium spp, Pseudomonas spp) the families Chlorobiaceae, Chromatiaceae, Ectoth- and certain fungi (Penicillum spp, Sordaria spp, Caliorhodospiraceae and RhodOspirillaceae have a ca- dosporium herbarium, Streptomyces spp) oxidize elepacity to oxidize H2S to varying degrees (Brune, mental sulfur and thiosulfate to sulfate, but this 1989). However, these microorganisms require light metabolic activity is much less than the thiobacilli as energy source and do not oxidize elemental sulfur species (Wainwright, 1978, 1984; Yagi et ai., 1971). very efficiently. The organisms Sulfolobus, Thermo- The two species of Thiomicrospira isolated until today plasma, Therrnothrix, Thermus and Thiobacillus ther- are not implicated in the process of metal leaching mophUica are not involved in the metal leaching from from sewage sludge. T. pelophila is very sensitive to sewage sludge because of their thermophilic nature acid and 7". denitdficans does grow if the oxygen (Brannan and Caldwell, 1980; CaldweU et al., 1976). concentration in the medium is very low (Kuenen and Certain bacterial species, which have a capacity to Touvinen, 1981). The thiobacilli therefore seem to be the only microorganisms, isolated until today, which possess the enzymatic system to rapidly oxidize eleTable 7. Metal composition in the sludges and their recommended mental sulfur as was observed during these investilevels gations in sewage sludge. Composition (mg/kg of dry sludge) The thiobcailfi are the organisms most often associCode Cd Cr Cu Mn Ni Pb Zn ated with sulfur compounds in the environment: A 5.0 87 215 933 28 il0 419 mining sites and elemental sulfur deposits (Hutchins D2 4.6 600 1607 327 154 161 517 et al., 1986; Laishley et al., 1988; Lundren and Silver, L2 7.7 321 603 1519 45 118 1205 1980; Reynolds et al., 1981). These microorganisms N 4.4 91 712 393 69 209 869 S3 7.9 98 2279 444 17 321 708 have also been found in diverse habitation: fresh water and sea water (Adair and Gundersen, 1969; Recommended 15 1000 1 0 0 0 1500 180 500 2500 level** Eashwar et a/., 1990; Fernandez and Vilda, 1988; *Flynn eta/. (1987). Turtle and/annasch, 1972) and different types of soil

of ceils is equivalent to 1.2 Stevens, 1991).

x 109

cells (Okereke and

107

Metal bioleachingfrom sewage sludge Table 8. Variation of pH, ORP and sulfate production after 5 days of microbial leaching pH Code A D2 L2 N $3

OPR (mV)

Sulfate (ms/I)

Initial

Final

Initial

Final

Initial

Final

% of sulfur oxidized

6.04 6.95 6.72 6.92 6.60

2.05 !.58 2.21 1.55 2.10

i 38 155 113 37 167

428 374 363 410 417

509 1512 606 781 874

3338 5250 4084 5198 3925

18.9 24.9 23.2 29.4 20.3

Table 9. Metal soluhilizat/on from the sludges after 5 days of microbial leaching % of soluhilization Code

CA

Cr

Cu

Mn

Ni

Pb

Zn

A D2 L2 N $3

83 88 84 90 87

19 33 (42)* 41 41 34

92 90 (46) 69 89 81 (57)

99 88 95 (2) 93 95

87 88 80 87 77

10 27 39 54 29

88 97 92

88 97

*Numbers in parentheses represent percentage solubilization required.

(Gore et al., 1986; Lee et al., 1987; Mahmoud et al., 1977). The presence of appreciable concentrations of thiobacilli in the sludges tested in the present study shows that this organism is included in normal indigenous sludge microflora. These results support the work of Fernandez and Vilda (1988) and Gore et al. (1986) who suggested that the presence of these microorganisms in fresh waters or in marine waters indicates a pollution load (domestic or industrial). The thiobacilli have also been isolated from municipal wastewater and sewage sludge by Hutchinson et al. (1969), Khalid and Malik (1987) and Sch6nborn and Hartman (1978), and they have also been found responsible for the corrosion in sewer pipelines (Milde et al., 1983; Sand, 1987). Three substrates have been proposed for the growth of thiobacilli in sewer lines: domestic wastes and detergents containing reduced sulfur compounds (Milde et al., 1983), the reduction of sulfates to produce H2S in sewer lines (Sand, 1987), and the production of volatile sulfur organic compounds resulting from the degradation of amino acids (Kadota and Ishida, 1972). The characteristics of the acidophilic species isolated in our studies indicate that ThiobaciUus thioox/dana is responsible for the acidification (pH from 4 to < 2) of all the sludges tested. The presence of several species of non-acidophilic thiobacilli in the sludge is responsible for the initial sludge acidification (pH

reduction from near 7 to 4.0). The less-acidophilic specieswhich reduces the p H from near 7.0 to 3.5-4.0 (group 4) seems to be the principalorganism for this activity.This group constitutesthe major population of less-acidophilic thiobacilli in all the sludge samples. Moreover, the growth'curves indicate that the concentration of the less-acidophilicthiobacilliis maximum at pH 3.5--4.0.According to Kelly and Harrison (1988), group 4 should be ThiobaciIIus thioparus. Therefore, this group plays an important role in the leaching process. These resultsare also in accordance with the resultsalready obtained on the mixed culturemetal leaching from sewage sludge (T. thiooxidana A T C C 55128 and T. thioparus A T C C 55127) (Blaiset aI., 1992a).The growth kineticsof the acidophilicand less-acidophilicspeciescorrespond to the growth of pure cultures of thiobacillinormally observed in the synthetic medium (Ahonen and Tuovinen, 1989; Blaiset aI., 1992a; W o o d and Kelly, 1985). This demonstrates that the sewage sludge constitutesa proper medium for the growth of these organisms. The presence of sulfur-oxidizing microflora in sewage sludge is potentiallyuseful for the removal of toxic metals found in sewage sludge. The solubilization yield of cadmium, copper, manganese, nickel and zinc obtained after 5 days' leaching are superior to the results obtained with the biological process using ThiobaciIIuaferrooxidana and ferrous sulfateas an energy source (Couillard and Chartier, 1991; Coulllard et aI., 1991; Tyagi and Tran, 1991; Tyagi et aI., 1990; W o n g and Henry, 1988). The possibility of solubilizationof chromium and lead is also better in the present process as compared to bioleaching with T. ferrooxidana. The solubilized metals in the leachate can be separated from decontaminated sludge solids by centrifugation or filtration. The metals in the leachate can be precipitated by neutralizing the leachate with lime. The decontaminated

Table 10. Growth kinetic parameters of the sulfur-oxidizing microflora during metal leaching

Code A D2 1.3 N $3

(dSO~.-/dt)t

V,~;

Leu.acidophilic (h-')

Acidophilic (h -I)

(gSO~4-/I x h)

(gS~4-/g cells x h)

0.079 0.091 0.091 0.104 0.084

0.067 0.072 0.078 0.079 0.071

0.055 0.075 0.060 0.061 0.068

1.70 2.27 1.39 2.28 2.34

• Maximum specific growth rate. tMaximum product formation rate. :~Maximum specific product formation rate.

J. F. BLAIS et al.

108

sludge solids can also be neutralized with lime before application to agricultural land. The proliferation of thiobacilli in nature is often associated with the severe environmental problems such as acid mine drainage (Lovell, 1983) and soil acidification (Bryant et al., 1983). However, this activity of microorganisms has already been commercially exploited or is under investigation (Hutchins et al., 1986; Kanagawa and Mikami, 1989). The results obtained in this study allow us to appreciate one other example of profitable use of these microorganisms. This process is presently under investigation at the pilot plant level for commercial exploitation. CONCLUSIONS This study has examined microbiological aspects essential for the development of a metal removal process from sewage sludge. The isolation and identification of the bacterial strains revealed that acid production is mused by a microbial flora composed of thiobacilli. The initial acidification of the sludge in the process of bioleaching is brought about by the growth of indigenous less-acidophilic thiobacilli followed by the growth of acidophilic thiobacilfi resulting in a pH reduction to near 2.0. The successive growth of less-acidophilic and acidophilic thiobacilli was observed in different types of sludge (secondary, aerobically and anaerobically digested) and with varying sludge solids concentration. The characterization of acidifying microflora showed that Thiobacillus thiooxidans is the only species belonging to acidophilic thiobacilli and Thiobacillus thioparus is the only important species (less-acidophilic) for initial sludge acidification. The other species may play a role in initial sludge acidification but only to a minor extent. The kinetic study on growth and sulfate production showed that the sludge type and sludge solids content do not significantly affect development of the thiobacilli. Acknowledgements---Sincere thanks are due to the Natural

Sciences and Engineering Research Council of Canada (Grants A4984 and STR 0100710), the Ministry of Education of Quebec (Grant FCAR 90-AS-9713) and Qu6bec University (Fonds de d6veloppement acad~nique du r~eau) for supporting this research. Thanks are due to S. Blackburn and N. Meunier for their technical assistance. REFERENCES Adair F. W. and Dundersen K. (1969) Chemoautotrophic sulfur bacteria in the marine environment. I. Isolation, cultivation, and distribution. Can. Y. Microbiol. 15, 345-353. Ahonan L. and Tuovinen O. H. (1989) Microbiological oxidation of ferroue iron at low temperatures. Appi. envir. Microbiol. $5, 312-316. APHA, AWV~A and WPCF (1989) Standard Methods for Examination of Water and Wastewater, 17th edition. American Public Health Association, Washington, D.C.

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