Trichloroethylene (TCE) removal in a single pulse suspension bioreactor

Journal of Environmental Management 74 (2005) 293–304 www.elsevier.com/locate/jenvman

Trichloroethylene (TCE) removal in a single pulse suspension bioreactor Viktor Volcˇ´ıka,*, Jaromı´r Hoffmannb, Jan Ru˚zˇicˇkab, Magda Sergejevova´c,d a

Faculty of Technology, Department of Physics and Material Engineering, Tomas Bata University in Zlı´n, namesti T.G. Masaryka 275, 762 72 Zlı´n, Czech Republic b Faculty of Technology, Department of Environmental Technology and Chemistry, Tomas Bata University in Zlı´n, namesti T.G. Masaryka 275, 762 72 Zlı´n, Czech Republic c Photosynthesis Research Centre, The Institute of Microbiology, Tomas Bata University in Zlı´n, Academy of Sciences, Trebon and The University of South Bohemia, Nove Hrady, Czech Republic d Institute of Landscape Ecology (ILE) of the Academy of Sciences, Tomas Bata University in Zlı´n, Nove Hrady, Czech Republic

Abstract This work describes TCE biotic removal in a single-pulse bioreactor under aerobic conditions. Activated sludge from a wastewatertreatment plant was used for inoculation of the cultivator. The experiment focused on a more detailed verification of microbial composition of mixed heterotrophic culture during pulsed phenol dosage. Attention was given to suppressing eucaryotic organisms, particularly yeasts and fungi, by the addition of cycloheximide. The TCE-removal capacity of the heterotrophic culture, described by kinetic tests, was dependent on pulsed phenol injection and on cyclic addition of phenol and TCE. Maximum TCE degradation was determined in a batch test. It was found that the addition of cycloheximide (an antibiotic against propagation and growth of fungi and yeast) increased the TCE degradation activity of the mixed microbial suspension. A certain residual amount of TCE remained in some of the experiments. q 2004 Elsevier Ltd. All rights reserved. Keywords: Trichloroethylene; Phenol; Cometabolism; Aerobic removal; Heterotrophic suspension

1. Introduction Trichloroethylene (TCE) and its metabolic intermediates belong to the most widespread and most problematic of groundwater pollutants. This compound is considered as xenobiotic, but is synthesized in a small quantity in natural conditions (Abrahamson et al., 1995; Dimmer et al., 2001). Its removal from the environment implies substantial problems and economic demands. Biodegradation is an interesting manner of TCE elimination. TCE-biodegradation under aerobic conditions can only be based on cometabolism, i.e. in the presence of another organic compound representing a primary carbon and energy source for the microorganisms. Some produced oxygenases possessing wider substrate specifity are capable of transforming a whole number of natural and synthetic substrates including TCE. In accordance with present knowledge, suitable primary

* Corresponding author. Tel.: C420 576 031 220. E-mail address: [email protected] (V. Volcˇ´ık). 0301-4797/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2004.09.001

substrates are, e.g. ammonia, methane, some aromatic compounds (phenol, toluene, ortho- and meta-cresol, cumene), propylene, propane (Ensley, 1991; Providenti et al., 1993) and dimethylsulphite (Takami et al., 1999). When comparing kinetics of TCE-biodegradation by aromatic-fed aerobic cultures, the maximum specific degradation rate and current transformation capacity (quantity of TCE degraded per quantity of biomass) were found for phenol-fed culture (Bielefeldt and Stensel, 1999; Lu et al., 1998). The composition of the microbial consortium and thereby also the TCE-removal ability is affected not only by the composition of the basic inoculum, but also by the mode of biomass feeding by phenol (Shih et al., 1996). Of the feeding variants tested (continuous and 3x discontinuous phenol dosage), the ‘fill-and-draw’ method with a 1-day cycle was the best (TCE degradation was 20!–100! faster, and there was the greatest representation of bacterial biomass and concentration of TCE-degrading microorganisms). Other feeding variants were characterized by a considerable quantity of fungal organisms present and thus low TCEremoval values (Shih et al., 1996). The fungi and yeast were

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Nomenclature TCE DCE C:N:P

trichloroethylene dichloroethylene ratio of dosage of carbon, nitrogen and phosphorus B1.B9 designations of kinetic tests of TCE biodegradation carried out with suspension from cultivator B

also observed in fill-and-draw cultivation, but in lower quantities. Primary substrates inducing synthesis of oxygenases are competitive inhibitors for TCE-oxidation during biodegradation because one enzyme is responsible for oxidation of both types of substrates (Chang and Alvarez-Cohen, 1995). Hence, if primary substrates are present at higher concentrations than required for stimulating biomass growth and thus adequate enzyme production, oxidation of cometabolite does not proceed or does so at a low rate. TCE intermediates or end-products (e.g. epoxide, chloral, CO) can abort its transformation (Cookson, 1995; Ishida and Nakamura, 2000). In addition, TCE at higher concentrations (approx. 40 mg lK1 TCE) may exhibit substrate toxicity to cultures producing oxygenases (Bielefeld et al., 1995; Folsom et al., 1990). This study is focused on verification and affection of microbial composition during “fill-and-draw” cultivation of mixed heterotrophic culture on phenol in greater detail, on constructing of equipment for observing TCE-degradation activity in a pulse mode, and on the study of TCEdegradation capacity dependently on phenol feeding and on repeated additions and quantity of TCE.

A2

VS MPA

designations of kinetic test of TCE biodegradation carried out with suspension from cultivator A volatile solids meat-peptone agar

continuously aerated and stirred with air. The volume of suspension in each pulse cultivator was 2.5 l. Exchange of suspension (always 500 ml) with simultaneous replenishment of phenol medium was carried out from the second day of feeding, once daily by the fill-and-draw method, resulting in a phenol dosage rate of 0.3 g lK1 per day. Hydrogen peroxide was added (0.5 ml of 30% solution of analytical purity to 2.5-l suspension) at the same time. The hydrogen peroxide concentration in the cultivator was 67 mg lK1. The pH in the cultivators was maintained at 8–8.5. The average age of the microbial suspension was 5 days. Cultivation proceeded at laboratory temperature, which varied from 22 to 24 8C. Ratios of basic nutrients C:N:P were 100:21:50 (possible exceptions mentioned in the text). Cycloheximide (50 mg. lK1) was added to cultivator B to suppress growth of eucaryotic organisms. Concentrations regularly observed were those of primary substrate (phenol), ammonia ions, pH, dissolved oxygen prior to and after fill-and-draw alternation, biomass dry substance, volatile solids; microscopic appearance of microbial suspensions was also kept under observation. Both cultivators were used solely for cultivation of microbial suspension, not for biodegradation of TCE.

2. Experimental 2.2. TCE-biodegradability tests 2.1. Semicontinuous cultivation of phenol utilizing micro-organisms Semicontinuous cultivation was performed using phenol as a sole carbon and energy source. Activated sludge from the municipal wastewater-treatment plant was decanted three times with drinking water before use for inoculation. Prior to inoculation, sludge was centrifuged (3000 rpm, 10 min) and resuspended in mineral medium to an initial solids content of 0.5 g lK1. The mineral medium was prepared from phosphate buffer pH 8.04 (solution of KH 2PO4 CK 2HPO 4CNa 2HPO 4), (NH 4) 2SO 4, CaCl2 , MgSO4, FeCl3 and a stock solution of trace elements (MnSO4CH3BO3CZnSO4C(NH4)Mo7O24 CCuSO4C Co(NO3)2) in accordance with the standard (CˇSN ISO 7827, 1994). Semicontinuous cultivation was always performed in parallel in two glass cultivators A and B which were

The microbial suspensions were tested for their TCEdegradation ability in an aerobic environment. Tests were conducted in TCE-bioreactors of approx. 640-ml volume. The bioreactors were filled with suspension and closed. Suspensions from the cultivators—used immediately without diluting or rinsing for degradation tests— contained a low quantity of phenol. The following operations (dosage of TCE solution, mineral medium for rinsing the dosage section of reactor, sample withdrawal) were executed by means of three-way valves. Two test variants differing in source of oxygen were employed: in the first, the reactor was completely filled (no headspace), available oxygen was dissolved in an aqueous medium and supplied by hydrogen peroxide, in the second setup (Fig. 1) pure oxygen was present above the surface of the suspension, and the bioreactor was not completely filled. The volume of the suspension was

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Fig. 1. Reactor for TCE-biodegradability tests. The volume of the reactor was approximately 640 ml. The reactor was not completely filled. The volume of suspension was 500 ml. Pure oxygen was present above the suspension. Position of valves: A, filling reactor with microbe suspension, filling with pure oxygen; B, dosing TCE solution, dosing mineral medium when withdrawing samples; C, withdrawal of samples, withdrawal of suspension.

approx. 500 ml. Usually 5 ml TCE solution at a concentration of 120 mg lK1 (actual concentration in the environment approx. 1.1 mg lK1 TCE) was dosed in, and the dosage section was subsequently rinsed with 20 ml mineral medium. During withdrawal of samples, 10 ml mineral medium was always dosed through the dosage section for rinsing the withdrawal section C5 ml for actually withdrawing sample. This arrangement enabled continuous addition of solutions or withdrawal of samples for TCE analyses. Abiotic tests were run concurrently with a suspension inactivated with mercuric chloride. An amount of 0.1 ml of 2.5% HgCl2 solution was applied to inactivate biotic tests. 2.3. Batch tests TCE-degradation ability of microbial suspensions was also tested in batch tests. These tests were run in a closed system under aerobic conditions. They were performed in 40-ml glass sample vials sealed with a silicon septum with Teflon film and polyethylene stopper. Suspension as well as resuspension was used in these tests. A resuspension was obtained by centrifuging the original suspension and resuspending it in a pure mineral medium. Twenty milliliters of suspension or resuspension were placed in vials. Abiotic tests of suspensions inactivated with 0.1 ml of 2.5% HgCl2 solution were performed simultaneously to

determine initial TCE concentration and possible abiotic losses. 2.4. Analysis of TCE Experiments were conducted by the ‘Purge and Trap’ method employing gas chromatography on a Hewlett Packard 5890 instrument following concentration in a Teckmar 2000 concentrator under these conditions: Vocarb 4000 sorbent, sample stripping time 11 min, trap drying 4 min, trap preheating 4 min, desorption 4 min, trap cleaning 4 min, trap preheating temperature 245 8C, desorption 260 8C, trap cleaning 245 8C. In the following GC analysis, conditions were as follows: capillary column Quadrex: 30 m!0.53 mm, 3-mm film (Quadrex Corp., USA), detection by flame-ionizing detector (FID), programmed temperature: starting 35 8C, final 200 8C, heating rate 4 8C minK1, starting time 10 min, final time 1 min, carrying gas (N2) flow rate 7.6 ml minK1, H2 flow rate 40 ml minK1, air flow rate 400 ml minK1. TCE identification and quantification was executed to external standards (prepared from a TCE solution in methanol, concentration 2 g lK1) by the calibration curve method. The sample volume was usually 5 ml. If TCE concentrations were greater than 1 mg lK1, a smaller sample was analysed or the sample was diluted. Concentrations were then calculated with respect to measured sample volume or dilution.

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2.5. Microbiological analyses

Table 1 Conditions of semicontinual cultivations

During cultivation, the composition of suspensions was subjected to microscopic observations (appearance and size of flocs, character of inter-floc liquid, presence of eucaryotic organisms—yeasts, fungi, protozoa). A NIKON microscope was used for observations. The magnifications were 100! and 400!. Floc size was determined by means of a Cirrus chamber. Concluding the last series, tentative identifications of dominant bacterial types were performed by Gram staining (Hucker, 1927), as well as determinations of aerobic bacteria by the plate count method on meat-peptone agar (MPA).

Number of series

2.6. Analytic procedures Phenol concentration was determined by photometry after the reaction of phenol with 4-aminoantipyrine and potassium persulphate in an alkaline medium (Standard CˇSN ISO 6439). Ammonia ions were determined following the reaction with Nessler’s reagent and Seignette salt (Standard CˇSN ISO 7150-1). Spectrophotometric measurements were executed with a photometer Spekol 11 (Karl Zeiss Jena, FRG). Concentration of dissolved oxygen or respiratory measurements of suspensions were performed using a membrane electrode Trioxmatic 300 with oximeter Oxi 359 (WTW, FRG). Biomass (expressed as suspended solids) was determined as an evaporation residue in crucibles annealed prior to use (550 8C, 2.5 h), in the following manner: 10 ml of suspension was evaporated at 90 8C and dried at 105 8C for 3 h. Content of mineral substances in the pure mineral medium was determined in the same way, and suspension dry matter corrected by this value (total solids TS). Crucibles were subsequently annealed at 550 8C for 2.5 h and ‘organic’ biomass fraction (volatile solids—VS in g lK1) was thus determined.

3. Results 3.1. Characterization of cultivation series—optimization of conditions Semicontinuous cultivation was successively performed in three series, running parallel in two cultivators. The conditions of particular cultivations are summarized in Table 1. Due to the insufficient buffer capacity of the low amount of phosphate buffer and the low pH of the phenol solution (pH 5), a considerable drop in the pH of cultivations during the first series occurred, as well as a subsequent excessive development of eucaryotic organisms (fungi, yeasts). Repeatedly increasing the suspension pH to a value of

1 2 3(*)

Conditions of cultivations Cultivator A

Cultivator B

100:21:6 100:21:50 100:21:50

100:5:6 100:5:50 100:21:50Ccycloheximide addition

Ratio of biogenous elements C:N:P was tested in the three cultivation series. The series number 3(*) was used for every TCE-biodegradation tests. Cycloheximide was added to the cultivation B—series number 3.

about 8.5 led to a partial decrease in the number of these organisms. In the second series a higher dosage of phosphate buffer was applied, the pH of the phenol stock solution was increased and the pH of the suspensions was continuously kept within 8–8.5. Very slow development of bacteria in cultivator B took place; their gradual disappearance and faster growth of eucaryotic organisms, particularly yeasts and fungi, were caused by a quantity of nitrogen four times lower than that in cultivator A. These possess phenol-degrading ability but have lower requirements with regard to the quantity of nutrients. Large zoogleotic bacterial flocs formed in the suspension with the higher nitrogen dosage (cultivator A) together with a very dense suspension of free bacteria in inter-floc liquid. Therefore, in the next (third) series the same ratio of basic nutrients in both parallel cultivations was chosen, and increased pH values were retained for both phenol solution as well as suspension. 3.2. Description of the third-series cultivations—differences Both parallel third-series cultivations differed merely in an addition of cycloheximide to cultivation B; cycloheximide together with the pH of the environment (pHO8) was able to reduce the growth of yeasts and fungi. According to literary data, cycloheximide inhibits protein biosynthesis in cells of some eucaryotic organisms (fungi, yeasts, some protozoic organisms) by bonding to their ribosomes (FLUKA catalogue; Obrig et al., 1971). Cycloheximide was added on days 5, 8 and 13 at a concentration of 50 mg lK1 and subsequently with daily regularity at a concentration of 10 mg lK1 (replenishing the consumed quantity in exhausted medium). The initial microscopic appearance of both suspensions (cultivators A and B) consisted of transparent inter-floc liquid with a small quantity of yeasts, fungi and diffuse flocs. Mixed cultures adapted only a little or virtually not at all to cultivation/feeding conditions. Phenol degraded slowly and during the first three days accumulated in the medium (up to 740 mg lK1). The concentration of phenol was generally less than 1 mg lK1 before its addition after adaptation of the suspension. The amount of volatile solids (VS) fluctuated from 500 to 900 mg lK1 and reached nearly 90% of the total

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Table 2 Characterization of suspensions from third series cultivations used for TCE biodegradability tests Test

Picture

Day of cultivation

Type of test

Suspension

Volatile solids VS (mg lK1)

Initial TCE concentration (mg lK1)

Final TCE concentration (mg lK1)

TCE removal in 24 h (%)

B2 B3 B4 A2 B6

Fig. 3 Fig. 3 Fig. 2 Fig. 2 Fig. 6

11 17 24 24 59

Kinetic test Kinetic test Kinetic test Kinetic test Kinetic test

B B B A B

718 668 556 652 446

B7

Fig. 6

59

Kinetic test

B

446

B8 B9 Bt B2

Fig. 7a Fig. 7b Fig. 5

66 66 31

Kinetic test Kinetic test Batch test

B B Suspension B Resuspension B

869 869 275 334

0.387 0.467 0.669 0.769 1.083 1.614a 1.300a 0.988 1.496a 1.394a 4.312 38.702 0.736 0.671

0.179 0.068 0.093 0.511 0.669 0.265 0.516 0.665 0.255 1.170 2.529 11.399 0.004 0.016

53.6 85.3 86.0 33.6 38.3 83.6 60.3 32.6 83.0 16.1 41.3 70.6 99.4 97.6

Suspension B was cultivated together with cycloheximide. a TCE concentrations after repetitive TCE additions.

solids (TS) in both cultivators. Suspensions from the third series were used for all TCE biodegradation tests. Characterization of suspensions used for TCE biodegradation kinetic or batch tests is given in Table 2. Suspensions A and B are from cultivators A and B, respectively. The TCE-degradation activity of the microbial suspension was also very low in the first days of both cultivations (the data are not given), as determined from TCE biodegradability kinetic tests on suspensions from cultivator A (14.8% drop in TCE concentration on day 4 of cultivation) and B (13.6% TCE-removal on day 6 of cultivation). The TCE-degradation efficiency of suspension B increased after 15 days to 85.3%.

A comparison of TCE-degradation of suspensions A and B on day 24 of cultivation showed that the presence of cycloheximide increased the TCE-degradation activity of suspension B (Fig. 2). Eighty-six percent of TCE was removed by suspension B4 in comparison with 33.6% by suspension A2 during a 24-h kinetic test. Differences in the microscopic appearance started to show from day 5 on, when the daily phenol dose was almost completely degraded. Repeatedly increased values of pH and dosage of cycloheximide resulted in almost complete disappearance of yeasts and fungi, zoogleotic flocs becoming dominant in suspension B. The number and size of bacterial flocs considerably increased and some of them began

Fig. 2. Comparison of TCE removal by different cultures (suspension A: test A2, suspension B with cycloheximide: test B4). Quantities of volatile solids (VS) were 652 and 556 mg lK1 in test A2 (-,-) and B4 (-B-), respectively. TCE degradation activity of suspension A (33.6% TCE removed during 24-h kinetic test) was smaller as compared with suspension B (86% TCE removed).

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Fig. 3. Increase in TCE-degradation activity of suspension B during cultivation (days 11: tests B2 and days 17: test B3). Quantities of VS were 718 and 668 mg lK1 in tests B2 (-,-) and B3 (-B-), respectively. Decreases in TCE concentrations achieved 53.6 and 85.3% after 22 h degradation.

forming floc agglomerates, while free rod-shaped bacteria were found in inter-floc liquid. Protozoic organisms predominantly present were infusoria and flagellata. Even when cycloheximide dosing had finished (day 44), overpropagation of yeasts and fungi and their dominance over the prevalent bacterial mass did not appear within the following 20 days. In suspension A there were excessive fungi, yeasts and protozoic organisms and a slight quantity of sludge flocs. Repeatedly increased values of pH proved to be effective as they led to almost complete disappearance of yeasts and fungi, zoogleotic flocs becoming dominant in the suspension. A very dense two-component suspension of free bacteria was formed in which rod-shaped bacteria were dominant. However, nearly at the end of cultivation (approx. day 50), great development of yeasts, fungi and protozoic organisms again occurred, especially of flagellata predominantly present in flocs. This picture did not considerably change up to the very end of cultivation (day 70). The cause of fluctuating degradation activity was an unstable microbial composition of the mixed culture. Varying microbial representation during cultivation was also noticeable visually. With suspension A, a change on day 28 of cultivation showed up by the fluorescent colour of the suspension potentially caused by the dominant presence of some bacteria of genus Pseudomonas. In suspension B at about the same time, readily sedimenting large flocs (several millimeter in size) were formed, and the suspension cleared up. Rotifers, infusoria and flagellata excessively propagated in flocs as well as in inter-floc liquid, and the quantity of mobile vacuolized rod-shaped bacteria increased. The ensuing change appeared as a red coloration in the suspension brought about by dominant microbial

representation of pigmenting GC cocci. Around day 40 of cultivation, disintegration of large flocs occurred and fine flocs less than 600 nm in size formed in the suspension. Inter-floc liquid was relatively transparent, with sporadic occurrence of free bacteria. A change in suspension colouring from red to yellow subsequently took place. This indicated a further change in the dominant organisms present. Total numbers of chemo-organotrophic aerobic bacteria at the close of the tests (day 65) determined during cultivation on meat-peptone agar were in the order of 108 CFU mlK1 in suspension A and about 107 CFU mlK1 in suspension B. 3.3. Characterization of TCE degradation ability of suspension B Gradual selection of micro-organisms in suspension B capable of degrading TCE and a corresponding increase in efficiency and rate of TCE-removal were proved by kinetic tests B2 and B3 (Fig. 3); after B-cultivations running 11, 17 and 24 days, decreases in TCE concentration of 53.6, 85.3 and 86% (B2, B3 and B4) were achieved after 24-h degradation. From the graphs showing the course of kinetic tests B2 and B3 (Fig. 3) and B4 (Fig. 2) it is obvious that after a certain time (1 day at most) a considerable slowdown in further TCE degradation took place, and the dependence of its concentration on time of degradation asymptotically approached a certain residual concentration (e.g. 64 mg lK1 TCE in test B3). The next picture (Fig. 4) shows results from the neutralised controls (where suspension was inactivated with mercuride chloride). The controls (CB2 and CB3) were carried out simultaneously with the tests B2 and B3. Decreases in TCE concentration of 10.6 and 8.2%

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Fig. 4. Results from the neutralised controls (suspension inactivated with 0.01% mercuride chloride). The controls (CB2 -&- and CB3 -C-) were carried out simultaneously with the tests B2 (-,-) and B3 (-B-). Decreases in TCE concentration achieved 10.6 and 8.2% (CB2 and CB3), respectively, after 24 h.

(CB2 and CB3) were achieved after approx. 24 h. From the comparison with results of the tests B2 and B3, it is obvious that TCE removal was in fact biodegradation rather than volatilization or other losses. Controls without added bioculture were not carried out. 3.4. Possible reasons for course of TCE-degradation The course of the above-mentioned kinetic tests could have been caused by a loss of cell enzyme activity (e.g. loss of oxygenases in absence of inductor, inhibition of

oxygenases by TCE-degradation intermediates or by cell waste products). If the presence of cell waste products did not mean irreversible inactivation of the enzyme system, then removal of initial liquid medium (from semicontinuous cultivation) by centrifuging, and washing of biomass with pure mineral medium followed with resuspension in fresh mineral medium should lead to an increased efficiency of TCE removal. Several batch tests with suspension and ‘resuspension’ from cultivation B proved the ability of the original suspension as well as the resuspension to efficiently remove TCE: 99.4 and 97.6% of TCE was degraded after

Fig. 5. Comparison of TCE removal by suspension and resuspension in batch tests. Suspension from reactor B (3rd series) was used for these tests—BtB2. The amounts of TCE degraded by suspension (–,–) and resuspension (-B-) were 99.1 and 97.6%, respectively. Differences in TCE degradation rates between suspension and resuspension were about 20% (cZ0.216 and 0.171 hK1, respectively).

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22 h (day 31 of cultivation). Differences in TCE-degradation rates between suspension and resuspension, based on kinetic constants of first-order formal kinetics, were about 20% (cZ0.216 and 0.171 hK1). The comparison of the tests carried out with suspension and resuspension is shown in Fig. 5. It is apparent from the results that the metabolites forming during cultivation on phenol obviously were not the cause of slow-down or even stopping of TCE-degradation. Another cause of incomplete TCE-degradation might have been insufficient oxygen for ensuring removal under aerobic conditions. The kinetic tests described so far were performed in two completely filled reaction vessels; available oxygen was dissolved in an aqueous medium. Respiration measurements of suspensions A and B gave values of specific respiration rates ranging from 3.0 to 4.2 mg O2 gK1 hK1. With kinetic tests, this meant complete exhaustion of oxygen after 3 h; the major part of the degradation test thus proceeded under conditions of oxygen deficit. For this reason further tests were performed in accordance with Fig. 1: pure oxygen (20–100 ml) was introduced above the surface of the microbial suspension at the onset of testing. This arrangement reliably guaranteed aerobic conditions throughout the whole test. Nevertheless, the residual quantity of TCE again remained undegraded. 3.4.1. Loss of oxygenases in the absence of inductor As the suspensions were in an endogenic state (without primary substrate), degradation of enzymes (catabolism), their use by cells as an energy source or their inactivation by intermediate products of TCE dissociation could also occur. This might have caused incomplete removal or low degradation of TCE.

In experiments B6 and B7, TCE and phenol (Fig. 6) were repeatedly added in the following kinetic tests. The goal was to find out if the cessation of TCE removal was caused by the toxicity of intermediate products or if undegraded residual TCE remained. These experiments were carried out with suspension B. The addition of phenol (10 mg lK1) at the beginning of test B6 as compared with test B7 achieved no considerable effect; their course was almost identical. TCE of 38.3% was removed after 20 h in comparison with 32.6% of TCE in test B7. After repeated doses of TCE (1 mg lK1)C10 and/or C50 mg lK1 of phenol, the course of degradation was different; in test B6 with 10 mg lK1 of phenol it was slower. More additions of phenol at 50 mg lK1 in both tests increased the efficiency of TCE removal. The results were not unambiguous. The final level of TCE removal was comparable but degradation stopped and 17% of the TCE remained irremovable. Repeated additions of 10 and 50 mg lK1 phenol and TCE produced differences between degradation of TCE in both tests. After the addition of 10 mg lK1 phenol, TCE was removed with efficiency comparable to that of the previous test but a certain amount of residual TCE was not degraded. An added 50 mg lK1 could lead to inhibited TCE-degradation; phenol, as primary substrate, is utilized by bacteria preferentially and could cause competitive inhibition. A residual TCE concentration remained in all tests; TCE was not completely removed. TCE was again degraded after being repeatedly added. On the basis of these results it may be assumed that alternating higher and lower additions of phenol could be employed for ensuring TCE degradation on a long-term basis. We are not able to consider what happened to the added phenol, because phenol was not measured. Therefore, we must

Fig. 6. Influence of repeated additions of TCE and phenol on the course of TCE degradation by suspension B. The amount of VS was 446 mg lK1. Repeated additions of phenol (10 and 50 mg lK1) led to induction of oxygenases and TCE-degradation. It appears an advantage for ensuring long-term TCE-degradation. (Descriptions by curves designate additions of TCECphenol in milligram per liter.)

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Fig. 7. Degradation of higher TCE concentrations by suspension B in tests B8 and B9. The amounts of VS were 869 mg lK1 in both tests. Initial TCE concentrations were 4.3 and 39 mg lK1 in the tests B8 (-,- a) and B9 (-B- b), respectively. Additions of phenol (50 and 300 mg lK1) after TCE degradation by 40 and 70% did not have a marked effect on further TCE degradation. Quantities of degraded TCE were 77 and 87% in tests B8 and B9. These results are comparable with the total degraded quantities in previous tests involving lower TCE concentration.

acknowledge the importance of this information in helping to interpret the results. Kinetic test B8 (Fig. 7a) proved the ability of the microbial suspension B to completely degrade TCE at concentrations of 4.3 mg lK1 (4.9 mg gK1 VS). When the initial TCE was degraded to 40% further degradation slowed down considerably. Repeated phenol additions of 50 and also 300 mg lK1 at the 24th and 50th hour, respectively, had no more effect as they did not lead to a more significant induction of the oxygenases necessary to ensure significant TCE-degradation. A mixed culture, obtained by the cultivation of activated sludge on phenol, was exposed to high concentrations of TCE up to 39 mg lK1 (45 mg gK1 VS; Fig. 7b). After repeated additions of phenol for sustaining induction of oxygenases the amount of TCE

degraded was 87%. This result is comparable to the total degraded quantity in the preceding tests with lower TCE concentration. Again, TCE was not completely degraded. The residual concentration of TCE was 13% from the initial concentration, although the ratio of substrate to TCE was 5 mg phenol/mg of TCE after 24 h of experiment. The transformation capacity was calculated as TcZ0.039 g TCE gK1 VS.

4. Discussion A mixed heterotrophic suspension with TCE-degradation ability can be obtained by cultivation on phenol-fed activated sludge (Shih et al., 1996; Shin and Lim, 1996).

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The degradation ability of the suspension can be directly affected by a suitable type of cultivation (Shih et al., 1996). Continuous cultivation is less suitable than pulse (batch) cultivation, as the excessive growth of fungal organisms and yeasts occurs, and the degradation rate is 100 times lower than in pulse cultivation (Shih et al., 1996). Fungal organisms and yeasts were also observed in the suspension during pulse cultivation (Shih et al., 1996). Aging of the suspension and the absence of growth substrate support their presence in a system. Addition of cycloheximide, which exerts an inhibitive effect on eucaryotic organisms and increased the pH 8, succeeded in markedly reducing the fungi and yeasts present in suspension and in increasing TCE degradation 2.5 times. A positive effect on increased TCE degradation and reduced quantity of fungi and yeasts is also produced by a shorter retention time and the permanent presence of growth substrate in the suspension, as well as a sufficient quantity of nitrogen in the medium. In contrast to mixed cultures, in the case of continuous cultivation of pure monoculture Pseudomonas cepacia G4, contamination of the suspension by fungi, yeasts and other organisms did not occur (Folsom and Chapman, 1991). A sufficient concentration of dissolved oxygen in the liquid phase is necessary for successful TCE degradation (Munakata-Marr et al., 1997). It was found that for the experiments described in the experimental part, the oxygen concentration in the liquid phase was sufficient for approx. 36 h depending on the quantity of biomass. Hence, it is necessary during TCE degradation to ensure the access of oxygen from a suitable source. It was found that the addition of hydrogen peroxide at a concentration of 67 mg lK1 lowers inoculum respiration rate by about 20% and TCE degradation by almost 10% (data not presented). A sufficient oxygen concentration during the 48–72 h degradation tests was ensured by adding pure oxygen to the gas phase of the reactor. Folsom and Chapman (1991) describe in their paper TCE degradation in a column reactor by bacteria P. cepacia G4 cultivated continuously in a chemostate. The authors of this paper do not mention or discuss oxygen balances in the reactor; the suspension in the chemostate was aerated. At an average 60-min retention time of TCE in the reactor, a minimum decrease in oxygen concentration may be assumed. It is supposed that dissolved oxygen is consumed for respiration by the suspension in a short time and its absence for TCE degradation is obvious. Despite this, TCE was degraded in the range of up to 50 mg dayK1. An average 60min retention time of TCE in a reactor is relatively short for TCE degradation in the range of 50 mg dayK1. MunakattaMarr et al. (1997) found that TCE degradation was almost completely stopped due to oxygen deficiency, and upon achieving oxygen balance TCE again underwent transformation. Data on the amount of TCE that causing toxic effects are relatively varied. A concentration of 40 mg lK1 did not cause inhibition or slowing down of the degradation

rate during degradation by the strain P. cepacia G4 (Folsom et al., 1990) or by a mixed culture obtained from surface water (Bielefeldt et al., 1995). In the case of a mixed culture of activated sludge cultivated on phenol, inhibition of TCE degradation at an initial concentration of 39 mg lK1 was not evident either. As opposed to this, a concentration of 42 mg lK1 exerted an inhibitive effect on the strain Ps. putida F1 (Wackett and Gibson, 1988). TCE was not completely degraded in any degradation test with mixed suspension of activated sludge cultivated on phenol. After repetitive addition, TCE was repeatedly degraded, therefore degrading enzymes were present in the system. Degradation again stopped before reaching complete transformation. The residual concentration was compared with the concentration in other experiments, where the initial concentration of TCE was approximately 1 mg lK1. A strong inhibitive effect on TCE degradation due to high TCE concentration (39 mg lK1) was not observed. A toxic effect of degradation intermediates on degrading enzymes or a lack of enzymes in the system was not taken into account. In contrast to our finding, Mars et al. (1998) described TCE degradation by a mixed culture of four strains of Pseudomonas during which a toxic effect of TCE or its metabolites on TCE-degrading strains occurred. After a certain time, the strain lacking degradation ability dominated the suspension. We did not succeed in finding the direct cause of cessation of TCE degradation prior to 100% transformation. We assume that at low TCE concentrations the diffusion of TCE molecules into cells stopped (Folsom et al., 1990). Diffusion was retarded by a low concentration gradient of TCE; restriction of transport due to the presence of cell metabolites did not occur (test suspension–resuspension, Fig. 4). Shurtliff et al. (1996) determined a suitable ratio of phenol and TCE, 5–10 mg phenol to 1 mg TCE. In accordance with this ratio, competitive inhibition did not occur and TCE degradation was supported (Bielefeldt et al., 1995; Shin and Lim, 1996). In the case of a mixed culture of activated sludge cultivated on phenol, the effect of simultaneous dosage of phenol which was tested in ratios of 5, 10 and 17 mg phenol to 1 mg TCE, was not evident. The course of degradation was identical to the test without added phenol. At a ratio of 50 mg phenol to 1 mg TCE, a slowdown in TCE degradation appeared together with a lower percentage of removed TCE, obviously due to competitive inhibition. The ratio of substrate to TCE in our experiment was 5 mg phenol/mg of TCE after 24 h of the experiment. This ratio should be optimal for degradation of TCE (Shurtliff et al., 1996; Shin and Lim, 1996), but the degradation of TCE was not complete. The transformation capacity (TcZ0.039 g TCE gK1 VS) was comparable with some published values (Chang and Alvarez-Cohen, 1995). Bielefeldt et al. (1995) found 10fold higher values of transformation capacity for bacteria growing on phenol, but our value may be underestimated, because the amount of TCE was measured in the liquid

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phase and we do not know the decrease in TCE in the gas phases.

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induction of enzymes by added phenol, however, produced little effect.

5. Conclusions

Acknowledgements

1. Discontinuous fill-and-draw cultivation of TCE-degrading bacteria may be recommended for its simplicity of operation and its capacity to provide inocula exhibiting TCE-degradation activity. 2. Activated sludge from the wastewater treatment plant can be used for inoculation. TCE degradability by a heterotrophic suspension was very similar in all cultivation series. The procedure of selecting microorganisms is not dependent on the quality of activated sludge and is not dependent on the time when inoculum was obtained from the wastewater treatment plant even though the composition of activated sludge is different on working days and/or weekends. TCE-degradation activity will be noticeable after 5–7 days of cultivation and will gradually reach a maximum in approx. 2 weeks. 3. Fluctuating degradation activity caused by unstable mixed cultures (changes in microbial representation) appeared during the course of cultivation. The instability was made evident by changes in microscopic appearance and/or visible changes in the colour of the suspension (ascribed to changes in dominant microorganisms). The employed mineral medium, type of cultivation and addition of antibiotic can significantly affect the composition of microorganisms in suspension. 4. The addition of cycloheximide during cultivation led to partially suppressed growth of yeasts and fungi and higher presence of TCE-degrading bacterial biomass. In the presence of cycloheximide, the bacterial suspension underwent faster adaptation and TCE-degradation activity increased more rapidly. Selection of microorganisms is positively affected by an increase in the pH of the phenol stock solution and continuous adjustment of suspension pH during cultivation (pHO8). 5. Complete TCE degradation was not achieved; biodegradation stopped upon achieving a certain degree of TCE conversion. This was not caused by an insufficient oxygen concentration or reversible inhibition (e.g. caused by cellular waste products in the aqueous medium from semicontinuous cultivation) nor particularly by a gradual loss of enzyme activity in the cells. Repeated addition of phenol to the residual TCE had no pronounced effect on further TCE degradation. 6. Repeated additions of lower and higher phenol doses (10 and 50 mg lK1) together with TCE (1 mg lK1) led to repeated induction of oxygenases and TCE degradation, which is advantageous for ensuring long-term TCE degradation. A certain residual TCE concentration always remained in the system. 7. The microbial culture was capable of partly removing even relatively high TCE concentrations; repeated

This work was carried out with the financial support of the Research Projects No. MSM 281100002 and partially by projects LN00A141 ‘Mechanism, Ecophysiology and Biotechnology of Photosyntesis’ and MSM123100004 of the Ministry of Youth, Education and Sports of the Czech Republic. References Abrahamson, K., Ekdahl, A., Colle´n, J., Pederse´n, M., 1995. Marine algae—a source of trichloroethylene and perchloroethylene. Limnology and Oceanography 40 (7), 1321–1326. Bielefeldt, A.R., Stensel, H.D., 1999. Biodegradation of aromatic compounds and TCE by a filamentous bacteria-dominated consortium. Biodegradation 1 (10), 1–13. Bielefeldt, A.R., Stensel, H.D., Strand, S.E., 1995. Cometabolic degradation of TCE and DCE without intermediate toxicity. Journal of Environmental Engineering 121 (11), 791–797. Chang, H.-L., Alvarez-Cohen, L., 1995. Transformation capacities of chlorinated organics by mixed cultures enriched on methane, propane, toluene or phenol. Biotechnology and Bioengineering 45, 440–449. Cookson, J.T., 1995. Bioremediation engineering—design and application, first ed. McGraw-Hill, New York, NY. Dimmer, C.H., McCulloch, A., Simmonds, P.G., Nickless, G., Bassford, M.R., Smythe-Wright, S., 2001. Tropospheric concentrations of the chlorinated solvents, tetrachloroethylene and trichloroethene, measured in the remote northern hemisphere. Atmospheric Environment 35, 1171–1182. Ensley, B.D., 1991. Biochemical diversity of trichloroethylene metabolism. Annual Review of Microbiology 49, 283–299. Folsom, B.R., Chapman, P.J., 1991. Performance characterisation of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4. Applied and Environmental Microbiology 57 (6), 1602–1608. Folsom, B.R., Chapman, P.J., Pritchard, P.H., 1990. Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates. Applied and Environmental Microbiology 56 (5), 1279–1285. Hucker, G.J., 1927. Further studies on the methods of Gram-staining. New York State Agricultural Experiment Station Technical Bulletin 1927;, 128. Ishida, H., Nakamura, K., 2000. Trichloroethylene degradation by Ralstonia sp. KN 1-10A constitutively expressing phenol hydroxylase: transformation products, NADH limitation and product toxicity. Journal of Bioscience and Bioengineering 89, 438–445. Lu, C.-J., Lee, C.M., Chung, M.-S., 1998. Comparison of trichloroethylene removal rates by methane- and aromatic-utilizing microorganisms. Water Science and Technology 7 (38), 19–24. Munakata-Marr, J., Mathenson, V.G., Forney, L., Tiedje, J.M., McCarty, P.L., 1997. Long-Term Biodegradation of Trichloroethylene Influenced by Bioaugmentation and Dissolved Oxygen in Aquifer Microcosms. Environmental Science & Technology. 31, 786–791. Obrig, T.G., et al., 1971. Journal of Biological Chemistry 246, 174. Providenti, M.A., et al., 1993. Selected factors limiting the microbial degradation of recalcitrant compounds. Journal of Industrial Microbiology 12, 379–395. Shih, C.-C., Davey, M.E., Zhou, J., Tiedje, J.M., Criddle, C.S., 1996. Effect of phenol feeding pattern on microbial community structure and cometabolism of trichloroethylene. Applied and Environmental Microbiology 8 (62), 2953–2960.

304

V. Volcˇ´ık et al. / Journal of Environmental Management 74 (2005) 293–304

Shin, H.S., Lim, J.L., 1996. Performance of packed-bed bioreactors for the cometabolic degradation of trichloroethylene by phenol-oxidizing microorganisms. Environmental Technology 17, 1351–1359. Shurtliff, M.M., Parkin, G.F., Weathers, L.J., Gibson, D.T., 1996. Biotransformation of trichloroethylene by a phenol-induced mixed culture. Journal of Environmental Engineering 7 (122), 581–589. Takami, W., et al., 1999. Evaluation of trichloroethylene degradation by E. coli transformed with dimethyl sulfide monooxygenase genes and/or cumene dioxygenase genes. Biotechnology Letters 21, 259–264.

Wackett, L.P., Gibson, D.T., 1988. Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1. Applied and Environmental Microbiology 54 (7), 1703–1708. ˇ SN ISO 7827, 1994. Standard C Standard CˇSN ISO 6439. Water quality. Determining monobasic phenols by spectrophotometry with 4-aminoantipyrine. ˇ SN ISO 7150-1. Water quality. Determining ammonia ions, Part Standard C 1-Manual spectrometric method. Catalogue of FLUKA Company.