Biodegradation of chlorinated aromatic hydrocarbons in slow sand filters simulating conditions in contaminated soil—Pilot study for in situ cleaning of an industrial site

Pergamon

0043-1354(94)00337-8

Wat. Res. Vol.29, No. 7, pp. 1663-1671,1995 Copyright © 1995ElsevierScienceLid Printed in Great Britain.All fights reserved 0043-1354/95$9.50+ 0.00

BIODEGRADATION OF CHLORINATED AROMATIC HYDROCARBONS IN SLOW SAND FILTERS SIMULATING CONDITIONS IN CONTAMINATED SOIL--PILOT STUDY FOR I N SITU CLEANING OF AN INDUSTRIAL SITE BERND ZACHARIAS,* ELKE L A N G t and HANS H. HANERT Microbiology Department, Technical University of Braunschweig, Konstantin-Uhde-Stra/~e5, 38106 Braunschweig,Germany (First received 7 July 1991; accepted 3 October 1991; received for publication 30 November 1994)

Abstract--13iodegradation of a mixture of chlorinated aromatic hydrocarbons (CAH) by a natural biocoenose of microorganisms was demonstrated in four semi-scale plants which served as models for a contaminated industrial site (concept of slow sand filters). The plants consisted of columns filled with contaminated soil taken from the ground of the spilled site and water storage containers that contained the contamiLnatedwater. The gas saturated water was continously recycled through the fixed bed columns. The gases supplied were either pure oxygen, air or nitrogen, with the nitrogen-supplied plant serving as a control simulating the microaerobic conditions in the natural habitat. A fourth plant was filled with sand instead of contaminated soil and supplied with air. It served only as a fixed bed and not as a reservoir for organic substrate. After 110 days, in all aerated plants more than 99% of the chlorobenzenes (CB), 96% of the chlorophenols (CP) and 94% of the adsorbable organic halogens (AOX) were removed. In comparison, only 82, 78 and 49% of these compounds were removedin the nitrogen-suppliedplant. Chloride ion concentrations in the aerated plants, measured as mineralization product of the AOX-concentrations, increased from 130 rag/1to 240 and 250 mg/l and coincidedwith the decrease of AOX-concentrations.Total cell counts of water and soil were approximately ten times higher in the aerated plants compared to the anaerobic or sandfilledplants. Microbial activity measured as respiration in the fixed beds showed the same profile. Key words--biodegradation,

chlorinated aromatic hydrocarbons, soil contamination, sand filter

INTRODUCTION Chlorinated aromatic hydrocarbons (CAH) are produced for use as pesticides such as DDT, Miiller and Lingens (1988), or the well-known wood preserving substance pentachlorophenol, Stanlake and Finn (1982) Intermediate substances in the production process often include several isomers of chlorinated phenols and benzenes. Microbial aerobic degradation of CAH was investigated by several researchers. Among others, Menke and Rehm (1992) investigated biodegradation of phenol and monochlorophencls in laboratory test systems, Baker and Mayfield (1980) focused on microbial decomposition of isomers of mono-, di- and trichlorophenols, Edgehill and Finn (1983) and Middeldorp et al. (1990) treated soil to remove pentachloropbenol, Reineke and Knackmuss (1984), Haigler et al. (1992) and *Author to whom all correspondence should be addressed at Sanitary Engineering Department, Technical University of Braunschweig. Pockelstra~ 4, 38106 Braunschweig, Germany. tPresent address: Ge:rmanCollectionof Microorganismsand Cell Cultures GmbH, Mascheroder Weg 16, 38116 Braunschweig, Germany.

Sander et al. (1991) isolated and described bacteria degrading several chlorinated benzenes. The biodegradation pathway of non-substituted phenol and benzene via cis-dihydrodiol, catechol and muconic acid is described by Cerniglia (1984) and Gibson (1977). Biodegradation pathways of the halogenated aromatic hydrocarbons are reviewed by Chaudhry and Chapalamadugu (1991) and Neilson (1990). The same enzymes that attack the nonchlorinated aromatic ring structure are responsible for the degradation of the halogenated derivatives. Chlorinated benzenes and phenols are hydroxylated to the corresponding catechol. Ring cleavage occurs either at the meta- or the ortho-position with the meta-cleavage often resulting in "dead end"metabolites. All quoted researchers describe biodegradation under aerobic conditions. Nicholson et al. (1992) describe anaerobic biodegradation of chlorinated phenols by methanogenic bacteria. Microbial decomposition of the mentioned pollutants is well established under aerobic conditions. However, all studies were done on a laboratory bench scale and none of the authors tried to transform these findings to technical or pilot scale experiments in order

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to assess the applicability o f the microbial degradation potential to polluted industrial sites. In this report, we demonstrate the biodegradability o f C A H spilled at an industrial site by using large scale pilot plants simulating the natural conditions. MATERIALS AND METHODS Origin and structure of soil During the work of a pesticide production plant intermediates of the manufacturing process had been introduced into the ground and subsequently contaminated the soil and the aquifer. The mixture of substances had mostly comprised chlorinated benzenes and chlorinated phenols and, to a minor part, isomers of hexachlorocyclohexane (HCH) and polychlorinated furanes and dioxins. A watertight layer was found at a depth of 6-10 m consisting of clay and silt with a varying thickness ranging from 0.4 to 4 m. The toxic substances, therefore, were concentrated above the sealing clay layer over the course of time by various spills on the site. In order to set up the pilot plants, at different places boreholes were set. The drill samples were thoroughly mixed, gastight sealed and stored in metal containers (volume approx. 0.3 m3). Contaminated water was directly taken from the ground and filled into the model plants. Model plant: construction, operation and rationale Four identical gastight plants were made out of high-grade steel (Fig. 1). Each plant consisted of a water storage container and a column with a fixed bed. The water was continously recycled through the columns. The water storage containers were each filled with 360 1of contaminated water, three columns were each filled with 40 kg of contaminated soil from the stored containers, one column was filled with sand (Fig. 2). The set up and the operating conditions for all plants are summarized in Table 1.

Before loading each plant was tested for gastightness by exposing a pressure of 3 bar. To avoid gas release while loading the water containers equal amounts of gas and water were exchanged continuously at a pressure of approx. 0.3 bar above atmospheric pressure. Although stripping losses were minimized while filling the containers, samples were taken and analyzed after having sealed the plants gastight and these data were set as start values for the following experiments. The water was saturated with the specific gas by permanently bubbling the gas through the water in the water storage tanks. Carbon dioxide produced during the process was removed by absorbing in soda-lye. Water samples were taken from a sample port in the outlet of a column, soil samples were taken from a sample port installed at each column. At the end of the experiment two plants were broken down and samples were taken from the fixed bed, the column and the storage container bottom to evaluate sorption and transport effects of the pollutants. Plants 2 and 3 were supplied with oxygen and air, respectively, in order to stimulate biodegradation, because this appeared to be the limiting factor. Plant 1 served as a control to show that oxygen-limited conditions are not appropriate for the degradation of the pollutants. The column of plant 4 served as uncontaminated fixed bed for the microorganisms. Electrochemical parameters In the beginning every 2-3 days and later on every 7 days, pH, temperature and oxygen concentration were monitored directly in the inlet and outlet of the columns. Oxygen concentration and temperature were measured with an oxygen meter OXI 92 from WTW, Weilheim, Germany, redox potential and pH were measured with electrodes from Metrohm, Herisau, Germany, or from lngold, Steinbach, Germany. Ion concentrations For control purposes of the physiological milieu the concentrations of dissolved iron, dissolved manganese,

2

E x--

10 [

0.15m

0.7m

Fig. 1. Semi-scale pilot plant comprising a fixed bed reactor and a water storage tank simulating the situation of a CAH contamination on an industrial site. Arrows indicate the direction of the water flow except for 10-14, which indicate the direction of the gas flow--l: fixed bed reactor (column), 2: valve, 3: measuring vessel at the column effluent, 4: measuring vessel at the column influent, 5: flow meter, 6: sample port for water samples, 7: pressure gauge, 8: water pumo, 9: water storage container, 10: ventilation, 11: connecting piece to ventilation system, 12: carbon dioxide aosorber, 13: gas bag filled with the specific gas, 14: air pump.

Chlorinated aromatic hydrocarbon biodegradation

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A B

C D Gravel (10-12 mm) Gravel (2-8 mm) Contaminated Soil Sand (0-2 ram)

Water-logged space

E Columns 1-3

Column 4

Fig. 2. Fixed bed reactors: technical details and different fixed beds---A: cover at column top with connecting piece, B: connecting piece with screw cap, C: sample port for glass slides with screw-cap, D: sample port with ball joint, E: bottom with connecting piece.

nitrate, nitrite, ammonia, phosphate and inorganic carbon in water were determir~ed periodically. Chloride ion concentrations were measured coulometrically with a Chloride Analyzer 926 (Corning, Halstad, U.K.). Concentration of inorganic carbon was determined according to DIN 38409-I~[7 and DEV Gl, Deutsches Institut fiir Normung (1979) and Fachgruppe Wasserchemie in der Gesellschaft Deutscher Chemiker (1989). All other ion concentrations were measured with testkits from Dr. Lange GmbH, Diisseldorf, Germany. The chloride ion concentration in soil was determined after extraction (10 g dry weight of soil in 25 ml of KNO3-solution, 0.2 mol/I) as described for water samples.

Organic substances Adsorbable organi!c halogens (AOX). The AOX-concentration in water was determined according to DIN 38409 H 14, Deutsches Instit'at fiir Normung (1985). The water was passed through two successively connected small activated carbon filter column~,;.Inorganic chloride was washed offwith nitrate-solution and the activated carbon containing the adsorbed organic substances was burnt at I I00°C. Organic chloride was converted to inorganic chloride and subsequently titrated coulometrically. Quality of adsorption was

calculated from the relation of bound organic chloride of the two filter columns. Extractable organic halogens. The method described in DIN 38409 H 8, Deutsches Institut fiir Normung (1984), for the determination of the EOX-concentration was slightly changed. 40 g wet soil were mixed with I ml H3PO4 (85%) and 40 g Na2SO4 and dried for 24 h. The mixture was then put into filters and extracted with 150 ml toluene for 2 h. The extract was burnt in an appliance by Wickbold and the mineralized chloride was determined turbidimetricly. 10 ml of the sample were mixed with 1 ml HNO3 (2 mol/l) and 0.5 ml reagent A (4.25 g AgNOj, 1 ml HNO3 conc. and 0.05 g NaCI dissolved in 100 ml water, heated to 70-80°C, filtered after cooling and stored at a dark place) and kept 10min in the dark. Following, the extinction at 365 nm was measured and the chloride ion concentration was calculated from chloride standards. Chemical oxygen demand (COD). COD concentrations in water were measured with test kits for water analysis from Dr. Lange GmbH. A solution of 150 mg glucose/I served as standard. The pollutants profile using gaschromatographical methods was done by Biocontrol Laboratory, Ingelheim, Germany.

Table 1. Plant operating parameters Hydraulic Delivery Flowrate RetentionTime Parameter Plant 1 Plant 2 Plant 3 Plant 4

Gas supply Nitrogen Oxygen Air Air

(l/h) 5 5 5 l0

(m/h) 0.283 0.283 0.283 0.566

(h) 6.4 6.4 6.4 3.2

Fixedbed Soil/Gravel Soil/Gravel Soil/Gravel Sand/Gravel

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Bernd Zacharias et al.

100 -

OXYGEN CONSUMPTION

,

(mg O2/h)

100 -

OXYGEN CONSUMPTION

(mg O ~

PLANT 2

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60"

B0-

40

40-

20

20-

O" i

0

20

r

40

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120

0

20

TIME (DAYS)

e0

80"

B0"

60-

40'

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20"

20-

0

0 40

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PLANT 4

80'

20

100

OXYGEN CONSUMPTION (rag O2/h)

loo

PLANT 3

0

80

TIME (DAYS)

OXYGEN CONSUMPTION (rag O2/h)

100

40

100

120

0

TIME (DAYS)

20

40

60

80

100

120

TIME (DAYS)

Fig. 3. Oxygen consumption, calculated from the difference of oxygen concentration in inflow and outflow of the fixed bed. Biological parameters

Chemicals

Total cell counts in soil and water were calculated from samples dyed with fluoresceine isothiocyanate (FITC) and viewed by u.v.-light microscopy. 10 #1 of a sample were exactly spread at 1 cm 2 of a glass slide, air dried and shortly heatfixed. Four parts of NaHCO3 (0.1 mol/i) and six parts of Na2COj (0.1 mol/l) were mixed with a small amount of FITC, filtered and applied to the slides. After 26q4) min the dye was washed offwith water and the slides were covered with ascorbate (I .25% in water) for 30~0 s and rinsed again. Before dying soil samples were treated with Na4PzO7 solution (2.8 g/l). 10 g soil were extracted with 5 ml Na4PzO7 solution for 20 min, the extract was collected, the extraction step was repeated and the extracts were combined. The final extract was treated as de-~ribed for water samples.

All chemicals had "p.A." quality and were obtained from Fluka Chemic AG, Buchs, Switzerland, or from Merck, Darmstadt, Germany. RESULTS

The oxygen c o n c e n t r a t i o n s of the water in the inlet o f the columns were approx. 8 a n d 12 mg/l for plants 3 a n d 4, respectively. In plant 2, which was supplied with pure oxygen, a n average c o n c e n t r a t i o n o f a b o u t 15 mg/1 was achieved a n d in the nitrogen-supplied p l a n t 1 the oxygen c o n c e n t r a t i o n remained mostly below 0.5 mg/l. Oxygen c o n s u m p t i o n in the columns

Chlorinated aromatic hydrocarbon biodegradation Table 2. Decreaseof extractableorganichalogens(EOX) in soil Plant 1 Plant 2 Plant 3 Time (soil/N2) (soil/O2) (soil/air) (days) (mg/kg) (mg/kg) (mg/kg) 0 804.88 770,50 611.22 6 n.d. 117.25 89.39 28 10.72 n.d. n.d. 69 41.41 n.d. n.d. 119 n.d. n.d. = not detectable(,: 10mg/kg).

of plants 2 and 3, calculated from the difference of oxygen concentrations in the inflow and outflow, reached maxima of 100 and 50 mg O2/h, respectively. On the contrary, in the microaerobic plant l no significant oxygen consumption could be determined and in plant 4 the oxygen consumption of 20 mg O.~/h at the beginning quickly ceased (Fig. 3). At the start, the EOX-concentrations in the contaminated soil of the columns l, 2 and 3 ranged

AOX (mgll)

COD (m0II)

180"~

'

40

from 600 to 800 mg chloride per kg soil (dry wt). After 1 week concentrations decreased to only 10-15% of the start concentrations (Table 2). No EOX-concentrations were detected in plant 4 at any time. AOX- and COD-concentrations of all plants are depicted in Fig. 4. Except for plant 4 which contained no contaminated soil, concentrations rose directly after beginning in all plants. In the nitrogen-supplied plant l concentrations rose until day 40 and stayed at a level of approx. 80 rag/1 COD and more than 17 mg/1 AOX, which is 51% of the maximal value. In the aerated plants 2 and 3 COD-concentrations were already decreasing after 7 days. The AOX-concentrations remained nearly constant for 40 days and decreased subsequently below 10% of their maxima. The correlating release of chloride ions is described in Fig. 5. In plants 2 and 3 release of chloride ions was detected until nearly all organic halogens (AOX) were used up. On the contrary, no chloride ion release could be detected in plant 4 and only little in plant I.

t20

PLANT 1

AOX (rag/l)

COD (moil) |

lool

100

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PLANT 2

/

-30

40

m~ 0

00-1

80

'°I

20

e0 ~

40"

80

0

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"10

-10

20

0

-80

80'

40

e0

100

80

0 120

80

40

GC)D (mill)

00

100

'0 120

TIME (DAYS)

TIME (DAYS)

AOX (m011)

180 ~

e0

40

120

GOD (rag/0

AOX (rag/l) PLANT 4

PLANT 3 100~

40

00

100" .80 80'

80

R0

e0-

'~'0

80'

40

40-

"10

10 80"

R 0

20

40

00

80

TIME (DAYS)

100

0 180

20

40

00

80

100

0 120

TIME (DAYS)

Fig. 4. Chemical :oxygen demand (COD) and adsorbable organic halogens (AOX), dissolved in water. Samples were taken out of the storage containers. Symbols: A COD, [] AOX.

Bernd Zacharias

1668 250 "

CHLORIDE (rag/I)

250

200 -

200

150 -

150

100 20

40

60

80

100

CHLORIDE (mg/I)

100

~ 0

et al.

120

TIME (DAYS)

20

40

60

80

100

120

TIME (DAYS)

Fig. 5. Chloride ion concentrations, dissolved in water. Samples were taken out of the storage containers. Symbols: [] plant 1, A plant 2, O plant 3, ~ plant 4.

Chloride ion concentrations in soil were nearly at the same level in all three plants filled with contaminated soil. At the start they ranged from 110 to 130mg C1/kg soil (dry wt). After 110 days approx. 50 mg C1/kg soil (dry wt) was found in each plant. The pollutant profiles of the water phases (Tables 3-5) that were done at the start and end of the experiment show that chlorinated benzenes made up the major part of the substance mixture. The start concentrations in all plants provided with contaminated soil exceeded 30,000/~g/1. More than 99% of the chlorinated benzenes were removed in all aerated plants whereas in the microaerobic plant 1 nearly 20% of the maximal concentration was still found after 110 days. In the aerated and substrate-provided plants 2 and 3, 97 and 98% of the chlorophenols were removed, contrasting a removal of only 78% in plant 1. pH increased continously from 7 to 9 during operation in all plants, since gaseous carbon dioxide was trapped in a CO_,-absorber. For equilibrium reasons bicarbonate was converted to dissolved carbon dioxide which was stripped. No differences in pH were detected in the inlet and outlet of the columns except for the columns of plants 2 and 3 between the days 20 and 40 (Fig. 6). The water temperature reached upper and lower values of 28 and 12°C, but mainly remained constant within a range 18-24°C. No correlation between temperature and microbial activity was found. Microbial populations were monitored as total cell counts in soil and water (Fig. 7). The population density in the aerated plants 2 and 3 was approximately ten times higher than in the nitrogensupplied plant 1 and in the substrate-deficient plant 4.

DISCUSSION

In the aerated plant 4 the AOX-concentration decreased right from the start. Due to the absence of organic halogens in the fixed bed no increase of AOX in the water phase occured. The rapid biodegradation showed that microorganisms are able to degrade the monitored substances without a significant lag phase if conditions are appropriate. The differences in biodegradation between the microaerobic and aerobic operated plants are demonstrated by the different AOX and COD levels which are significantly lower in plants 2 and 3 compared to plant 1 as well as in the rapid decrease of the AOX-concentrations after 40 days. AOX-concentrations in plants 2 and 3 turned out to be rather similar to each other. The higher oxygen supply in plant 2 had no effect on biodegradation. After 40 days the substances measured as AOX were mostly degraded. On the contrary, the microaerobic milieu in plant 1 was insufficient to support microbial degradation of the pollutants to this extent. Only 49 % of the AOX could be removed after the monitored time range. However, about 80% of the analysed chlorinated benzenes and phenols were removed. The original substances were obviously degraded but intermediates, still chlorinated, remained recalcitrant and explain the AOX removal of only 49%. It can be assumed from these findings that still less oxygen concentrations in plant 1 would have left the pollutants even more undegraded. The rapid decrease of the EOX-concentrations in the soil of the columns 1, 2 and 3 during the first week (Table 2) was not only due to biodegradation but mostly to saturation of the water phase and to mobilization of fine particles highly covered with

Chlorinated aromatic hydrocarbon biodegradation CAH. As a consequence, the AOX-concentrations of t h e s e t h r e e plants~ m e a s u r e d in t h e w a t e r , i n c r e a s e d d u r i n g t h e first d a y s . A t t h e e n d o f t h e e x p e r i m e n t s the plants were investigated for traps of pollutants. N o s i g n i f i c a n t a n a o u n t s o f p o l l u t a n t s were f o u n d d e p o s i t e d a n y w h e r e , e i t h e r in t h e c o l u m n s o r in t h e water storage tanks. T h e different eJ~ects o f a e r o b i c o r m i c r o a e r o b i c o p e r a t i n g c o n d i t i o n s were also reflected b y t h e different c o n c e n t r a t i o n s o f i n o r g a n i c chloride. A f t e r t h e e q u i l i b r i u m b e t w e e n soil a n d w a t e r h a d b e c o m e constant no further increase of chloride ions dissolved in w a t e r were m o n i t o r e d in p l a n t I (Fig. 5). T h e overall i n c r e a s e o f 35 m g c h l o r i d e / l w a s s m a l l c o m p a r e d to t h e m o n i t o r e d i n c r e a s e o f m o r e t h a n 100 m g chloride/1 in t h e a e r a t e d p h m t s 2 a n d 3 w i t h i n 60 d a y s o f operation. These findings correlate with the AOXc o n c e n t r a t i o n s in p l a n t s 2 a n d 3 w h i c h d e c r e a s e d w i t h i n t h e s a m e t i m e p e r i o d o f 60 d a y s b e l o w 1 0 % o f their maxima. In plant 4 no significant increase of dissolved inorganic chloride was monitored because of the minimal concentration of organic halogens supplied. T h e m i c r o b i a l p o p u l a t i o n s o f all p l a n t s a r e d e s c r i b e d in Fig. 7. Cell c o u n t s in t h e a e r a t e d p l a n t s 2

Table 3. Pollutants prc,file of the water phase after equilibrium between soil and water was achieved; monitored 6 days after the start of the recirculation of the water phase

Parameter Chlorinated benzenes: Mono-CB 1,2-Di-CB 1,3-Di-CB 1,4-Di-CB 1,2,3-Tri-CB 1,2,4-Tri-CB 1,3,5-Tri-CB 1,2,3,4-Te-CB 1,2,3,5 + 1,2,4,5-Te-CB Penta-CB Hexa-CB Sum CB: Chlorinated phenols: 2-Mono-CP 3-Mono-CP 4-Mono-CP 2,3-Di-CP 2,4 + 2,5-Di-CP 2,6-Di-CP 3,4-Di-CP 3,5-Di-CP 2,3,4-Tri-CP 2,3,5-Tri-CP 2,3,6-Tri-CP 2,4,5-Tri-CP 2,4,6-Tri-CP 3,4,5-Tri-CP 2,3,4,5-Te-CP 2,3,4,6-Te-CP 2,3,5,6-Te-CP Penta-CP Sum CP: Sum HCH:

Plant 1 Plant 2 Plant 3 Plant 4 (soil/N2) (soil/O:) (soil/air)(sand/air) (.ug/l) (~g/l) (/zg/l) (/tg/l) 76 358 815 591 6150 22800 1170 392 495 10 1 32858 7 18 9 4 119 4 320 10 33 21 56 147 17 3 17 34 4 19 842 308

9 113 6 308 855 734 520 652 6130 5520 22600 21300 1181 1010 375 278 486 337 9 4 1 n.d. 3 2 1 7 2 30256 7 24 10 3 103 4 231 8 33 20 52 132 15 3 13 28 4 19 709 290

7 26 12 3 90 3 296 7 26 17 44 112 13 2 11 30 3 17 719 289

240 220 403 8 262 2190 52 351 62 20 1 3809 16 31 18 3 140 2 440 6 15 14 36 141 7 2 7 6 2 2 888 239

CB = chlorinated benzenes, CP = chlorinated phenols, HCH= bexachlorocyclohexane, n.d. = not detectable.

1669

Table 4. Pollutants profile of the water phase at the end of the experiment (after 110 days recirculation) Plant 1 Plant 2 Plant 3 Plant 4 (soil/N2) (soil/O,.) (soil/air)(sand/air) (#g/I) (pg/I) (,ug/l) (,ug/I)

Parameter Chlorinated benzenes: Mono-CB 1,2-Di-CB 1,3-Di-CB 1,4-Di-CB 1,2,3-Tri-CB 1,2,&Tri-CB 1,3,5-Tri-CB 1,2,3,4-Te-CB 1,2,3,5 + 1,2,4,5-Te-CB Penta-CB Hexa-CB Sum CB:

1 2 14 5 2350 2580 358 352 378 12 1 6053

n.d. n.d. n.d. n.d. I 1 47 1 17 11 2 80

n.d. n.d. n.d. n.d. 2 1 34 1 10 6 2 56

n.d. n.d, n.d. n.d. I 1 5 2 3 2 n.d. 14

4 1 1 4 28 9 4 2 10 11 35 40 14 1 n.d. 14 2 4 184 210

n.d. n.d. n.d. 1 1 n.d. 1 n.d. 1 1 1 8 n.d. n.d. n.d. 7 n.d. 3 24 31

1 n.d. n.d. 1 1 n.d. 1 n.d. 1 o.d. 1 5 n.d. n.d. 1 n.d. n.d. n.d. 12 33

n.d. n.d. n.d. n.d. 1 n.d. n.d. n.d. n.d. n.d. n.d. 1 n.d. n.d. n.d. n.d. n.d. n.d. 2 9

Chlorinated phenols: 2-Mono-CP 3-Mono-CP 4-Mono-CP 2,3-Di-CP 2,4 + 2,5-Di-CP 2,6-Di-CP 3,4-Di-CP 3,5-Di-CP 2,3,4-Tri-CP 2,3,5-Tri-CP 2,3,6-Tri-CP 2,4,5-Tri-CP 2,4,6-Tri-CP 3,4,5-Tri-CP 2,3,4,5-Te-CP 2,3,4,6-Te-CP 2,3,5,6-Te-CP Penta-CP Sum CP: Sum HCH:

CB = chlorinated benzenes, CP = chlorinated phenols, HCH= hexachlorocyclohexane, n.d. = not detectable).

a n d 3 a r e a p p r o x i m a t e l y o n e m a g n i t u d e h i g h e r t h a n in the other plants. In plant 1 oxygen was the limiting f a c t o r , in p l a n t 4 o r g a n i c s u b s t r a t e w a s t h e l i m i t i n g growth factor. T h e h i g h e s t o b s e r v e d cell c o u n t s c o i n c i d e d w i t h t h e m a x i m a l o x y g e n c o n s u m p t i o n r a t e s (Fig. 3). T h e

Table 5. Sums of the chlorinated substances and percentage of removals Parameter

Plant 1 Plant 2 Plant 3 (soil/N2) (soil/O2) (soil/air)

Chlorinatedbenzenes (CB): Start (pg/l) 3 2 8 5 8 32172 End (#g/I) 6053 80 Removal (%) 81.58 99.75

Plant 4 (sand/air)

30256 56 99.81

3809 14 99.63

Chlorinated phenols (CP): Start (pg/I) 842 End (pg/l) 184 Removal (%) 78.15

709 24 96.61

719 12 98.33

888 2 99.77

Hexachlorocyclo-hexanes Start (ug/l) End (~ug/l) Removal (O/o)

290 31 89.31

289 33 88.58

239 9 96.23

Adsorbable organic halogens (AOX): Maximum (mg/1) 34.50 15.25 End (mg/l) 17.72 0.79 Removal (%) 48.64 94.82

14.59 0.69 95.27

4.97 0.26 94.77

(HCH): 308 210 31.82

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Bernd Zacharias et al.

~H

10

pH

10

l

PLANT2

f

PLANT3

J f

,

i

i

,

i

20

40

60

80

100

120

0

i

i

i

i

t

20

40

60

80

100

TIME (DAYS)

120

TIME (DAYS)

Fig. 6. pH-values of influents and effluents of the fixed bed reactors of plant 2 and plant 3. Symbols: [] pH (influent), ~ pH (effluent).

highest pH-differences were measured in the inlet and outlet of columns 2 and 3 between days 20 and 40 due to respiration and subsequent acidification by carbon dioxide production as well as high biodegradation rates of AOX with hydrogen chloride production. After 50-60 days, population density as well as oxygen consumption rates decreased, thus, at the same time dissolved organic halogens (AOX) also decreased to minimal concentrations. Since oxygen was still available, organic substances must have become rate-limiting. The organic matter monitored as COD which remained constant after 40-50 days at a level of

1000

approx. 40 mg/l in plant 2 and 35 mg/1 in plant 3 obviously did not serve as organic substrate for the biomass. It is well known that bacteria have the potential to break down a great diversity of organic chlorines but most investigations have concentrated on laboratoryscale experiments so far. The data presented here show that the application to pilot-scale plants that simulate the original contaminated ground will lead to a high elimination of CAH. This study is meant to be one step towards the direct in situ treatment of contaminated soils and waters.

106 cells per ml

106 cells per g soil (dry wt)

SOIL

1ooI

WATER

100

10

1

J

20

40

i

60

80

TIME (DAYS)

100

0,1

120

140

~

0

20

40

60

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120

140

TIME (DAYS)

Fig. 7. Total cell counts of bacteria attached to the soil surface of the column fixed beds or suspended in the container water. Cells were dyed with fluorescine isothiocyanate (FITC) and made visible under u.v.-light. Symbols: [] plant 1, A plant 2, O plant 3, W plant 4.

Chlorinated aromatic hydrocarbon biodegradation

REFERENCES Baker M, D. and Mayfield C. I. (1980) Microbial and non-biological dex:omposition of chlorophenols and phenol in soil. War. Air Soil Pollut. 13, 411--424 Cerniglia C. E. (1984) Microbial transformation of aromatic hydrocarbons. In Petroleum Microbiology (Edited by Atlas, R.), pp. 99-]127. Macmillan, New York. Chaudhry G. R. and Chapalamadugu S. (1991) Biodegradation of halogenated organic compounds. Microbiol. Rev. 55, 59-79 Deutsches Institut fiir Normung (DIN) (1979) Summarische Wirkungs- und Stoffkenngrrflen (Gruppe H), Bestimmung der S/lure- und Basekapazit/it, DIN 38409 H7, DIN, Berlin. Deutsches Institut f'tir Normung (DIN) (1984) Summarische Wirkungs- und Stottkenngrrflen (Gruppe H), Bestimmung der extrahierbaren organisch gebundenen Halogene (EOX), DIN 38409 H8. DIN, Berlin. Deutsches Institut fiir Normung (DIN) (1985) Summarische Wirkungs- und Stoflkenngrrflen (Gruppe H), Bestimmung der adsorbierbaren organisch gebundenen Halogene (AOX), DIN 38409 H14. DIN, Berlin. Edgehill R. U. and Finn R. K. (1983) Microbial treatment of soil to remove pentachlorophenol. Appl. Environ. Microbiol. 45, 1122--1125, Fachgruppe Wasserchemie in der Gesellschaft Deutscher Chemiker (Eds) (1989) Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung (DEV), G l: Bestimmung der Summe des gelrsten Kohlendioxids. Fachgruppe Wasserchemie in der Gesellschaft Deutscher Chemiker, Verlag Chemie, Weinheim. Gibson D. T. (1977) Microbial transformations of aromatic pollutants. In Proc~ Int. Syrup. on Aquatic Pollutants

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(Edited by Hutzinger O.), pp. 187-204. Pergamon Press, Oxford. Haigler B. E., Pettigrew C. A. and Spain J. C. (1992) Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JSI50. Appl. Environ. Microbiol. 58, 2237-2244. Menke B. and Rehm H. J. (1992) Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells ofAlcaligenes sp. A7-2. Appl. Microbiol. Biotechnol. 37, 655-661. Middeldorp P. J. M., Briglia M. and Salkinoja-Salonen M. S. (1990) Biodegradation of pentachlorophenol in natural soil by inoculated Rhodococcus chlorophenolicus. Microb. Ecol. 20, 123-139. Miiller R. and Lingens F. (1988) Der mikrobielle Abbau yon chlorierten Kohlenwasserstoffen. g w f . Wass. Abwass. 129, 55-60. Neilson A. H. (1990) The biodegradation of halogenated organic compounds. J, appl. Bacteriol. 69, 445-470. Nicholson D. K,, Woods S. L., Istok J. D. and Peek D. C. (1992) Reductive dechlorination of ehlorophenols by a pentachlorophenol-acclimated methanogenic consortium. Appl. Environ. Microbiol. 58, 2280-2286. Reineke W. and Knaekmuss H. J. (1984) Microbial metabolism of haloaromatics: isolation and properties of a chlorobenzene-degrading bacteria. Appl. Environ. Microbiol. 47, 395-402 Sander P., Wittich R.-M., Fortnagel P., Wilkes H. and Francke W. (1991) Degradation of 1,2,4-trichioro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Appl. Environ. Microbiol. 57, 1430-1440. Stanlake G. J. and Finn R. K. (1982) Isolation and characterization of a pentaehlorophenol-degrading bacterium. Appl. Environ. Microbiol. 44, 1421-1427.