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Operation parameters affecting the survival of genetically engineered microorganisms in activated sludge processes
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Pergamon
O043-1354(93)EOO29-R
War. Res. Vol. 28, No. 7, pp. 1667-1672, 1994 Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/94 $7.00+ 0.00
OPERATION PARAMETERS AFFECTING THE SURVIVAL OF GENETICALLY ENGINEERED MICROORGANISMS IN ACTIVATED SLUDGE PROCESSES MASANORIFUJITA*~, MICHIHIKOIKE and KAZUYAUESUGI Department of Environmental Engineering, Osaka University, Yamadaoka, Suita, Osaka 565, Japan (First received April 1993; accepted in revised form November 1993)
Abstract The survival of genetically engineered microorganisms (GEMs) harboring recombinant plasmid pBHS00, containing catechol 2,3-oxygenase encoding gene, in model activated sludge processes was investigated. Escherichia coil C600 (pBH500) and Pseudomonas putida BH(pBH500) were inoculated into activated sludge and cultivated in the fill and draw (FD) and continuous flow (CF) systems under different conditions. In both systems, the populations of introduced GEMs declined rapidly during the initial period (5-10 days for the FD system and 5-15 days for the CF system), after which they remained relatively stable. In the FD system, the larger the inoculum size, the higher the population level at which the GEMs remained stable. However, in the CF system, repeated inoculation did not improve the survival ofP. putida BH(pBH500). The sludge retention time (SRT) affected the survival of GEMs considerably in both systems; as the SRT of the system was decreased, the survival populations of GEMs increased. The presence of phenol, which can support the growth of P. putida BH(pBH500), did not influence its survival. Key words--survival of genetically engineered microorganisms, activated sludge process, sludge retention time, inoculum size, fill and draw system, continuous flow system, Pseudomonas putida, Escherichia coli, selective pressure of ecosystem
INTRODUCTION The potential for the improvement of wastewater treatment by using genetically engineered microorganisms (GEMs), especially in the effective removal of xenobiotic or toxic compounds, has been recognized by many researchers (Fujita et al., 1991; Kobayashi, 1984; McClure et al., 1991a). GEMs capable of degrading a wide variety of xenobiotic compounds or showing enhanced degradation activities have already been constructed (Fujita et al., 1993; Kellog et al., 1981; Chapman, 1988; Timmis et al., 1988). However, before this approach can be realized, the problem of the ecological stability of GEMs must be solved. Ecological stability refers to the length of time that the GEMs can survive and express their useful activities in the mixed microbial flora of wastewater treatment processes. If a G E M has a low ecological stability, it may soon disappear from the process and the treatment efficiency will thus be little improved, even if a G E M with a high xenobiotic compound degradation rate is constructed. The survival of introduced bacterial strains, including GEMs, in the mixed microbial flora of natural environments or microcosms, such as soils, sediments, sewage and pond/lake water has been dealt with in numerous studies (Stotsky and Babich, 198@
*Author to whom all correspondence should be addressed.
As for the survival in wastewater treatment processes, McClure et al. (1991 b, 1989) investigated the survival of natural and genetically engineered bacteria capable of degrading 3-chlorobenzoate in a laboratory-scale activated sludge unit, and pointed out the importance of choosing strains that are well-adapted to the environmental conditions for the successful use of microbial inoculation. However, to date, little is known about the mechanism of GEM survival in wastewater treatment processes. This study was designed to investigate the survival of two GEMs, Escherichia coil C600(pBHS00) and Pseudomonas putida BH(pBH500) (Fujita et al., 1991, 1993) harboring a recombinant plasmid in both fill and draw (FD) and continuous flow (CF) model activated sludge systems, which simulated fed-batch/ batch and conventional activated sludge treatment processes, respectively. An attempt was made to determine some of the operational parameters which might influence the survival of GEMs.
METHODS
MATERIALSAND Bacterial strains and plasmid E. coli C600 (Appleyard, 1954) and phenol and benzoateutilizing P. putida BH were used as host strains of recombinant plasmid pBHS00 (Fujita et al., 1991). Recombinant plasmid pBH500 was constructed by inserting the 5.65 kbDNA fragment containing catechol 2,3-oxygenase (C230), . which catalyses the conversion of catechol into 2-hydroxymuconic semiaidehyde (2-HMS), encoding gene (pheB)
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MASANORI FUJ1TA et al.
from the chromosome of P. putida BH, into a broad host range vector plasmid pKT230 (Bagdasarian et al., 1973). This plasmid gives the host strains resistance to streptomycin (Sm) and the constitutive expression of pheB. Colonies expressing pheB can be identified by spraying with 0.I mM catechol solution. Positive colonies quickly turn yellow due to the formation of 2-HMS. The phenotypes of the bacterial strains and plasmid pBH500 are listed in Table 1.
Media Both GEMs, E. coli C600(pBH500) and P. putida BH(pBH500), were routinely maintained on CGY medium (Pike et al., 1972) supplemented with Sm, and grown in modified L broth (LM broth) containing 10g of Bacto peptone, 5 g of Bacto-yeast extract and 5 g of NaC1 in I liter of deionized water (pH = 7.4) with Sm at 28~'C on a rotary shaker (120 rpm). CGY medium was also used for enumeration of total heterotrophic bacteria. Counts of the GEMs were carried out with the selective media as follows. E. coil C600(pBH500) was enumerated by using deoxycholate agar (Eiken Chemical Co., Tokyo, Japan) for isolation of enteric bacteria with 100 mg/l Sm and P. putida BH(pBH500) by benzoate medium containing l g of K~HPO4, 1 g of (NH4)2SO 4, 0.1 g of MgSO4, 0.02g of FeC13, 0.1g of NaC1, 0.1g of CaCl 2, 0.5g of sodium benzoate and 15 g of agar (pH = 7.4) in 1 liter of deionized water with 50 mg/l Sm. Model activated sludge systems The activated sludge used in this study had been acclimated to synthetic wastewater containing 0.2 g of meat extract, 0.3 g of peptone, 0.05 g of urea, 0.015 g of NaCI, 0.007 g of KC1, 0.007 g of CaC12, 0.005 g of MgSO4 and Na 2HPO4 in 1 liter of tap water for more than 2 years by the fill and draw technique. Two model activated sludge systems were constructed and operated as follows. Fill and draw (FD) system. Activated sludge treatment by the FD system was carried out by shake culture. GEMs were grown overnight, pelleted by centrifugation (10,000g, 15 min) and added to 100 ml of the activated sludge (MLSS conc. = approx. 2000 mg/l). The sludge was cultivated in a 300-ml Erlenmeyer flask at 25°C on a rotary shaker (100 rpm). After 21 25-h cultivation, a specific volume of waste sludge was withdrawn to control the sludge retention time (SRT) of the system, and the rest was settled for 30 min, The supernatant (85-90% of the total volume) was then discarded, and the sterilized double-strength synthetic wastewater above was added up to 100 ml. These operations were repeated daily. Continuous flow (CF) system. The CF system consisted of an aeration tank (working volume = 5 l) with a liquid overflow to a settling tank (working volume = 1 liter), the sludge being returned to the aeration tank (4,31/day). Double-strength synthetic wastewater was fed continuously (5 1/day) to the aeration tank. Aeration was conducted at 0.4 vvm of air with an air pump and diffuser attached at the
Table I. Bacterial strains and plasmid used in this study Strain or plasmid Phenotype* E. coli C600 thi ,leu ,thr ,Sm ~ P. putida BH Ben+, Phe+, Sm' Isolated from activated sludge pBH500
Sm~, C230 Constructed by inserting 5.65kb-DNA fragment from the chromosomeof P. putida BH into pKT230 *Abbreviations:requirementsfor thiamine(thi- ), leucine(leu ) and threonine(thr- ); growth on benzoate(Ben+ ) and phenol(Phe+ ); resistant to (Sm~)or sensitiveto (Sin') streptomycin;constitutive expression of pheB (C230).
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Fig. 1. Survival of P. putida BH(pBH500) in the FD activated sludge system (SRT = 20 days). Viable counts of total heterotrophic bacteria ( 0 ) and the GEM (©) are shown. bottom of the tank, in which the temperature was maintained at 25°C. Waste sludge was withdrawn from the aeration tank. GEMs were harvested in the same manner as described above and added to the aeration tank. The feeding ofwastewater was stopped after the inoculation for 1 or 24 h (after the first inoculation on day 0, the feeding was stopped for 24 h).
Enumeration of GEMs Activated sludge samples withdrawn were harvested by centrifugation (10,000g, 10rain) and resuspended in the same volume of 5 mg/l s,~dium tripolyphosphate solution (tpp.). A 50 ml sample diluted 10-fold with tpp. was treated with a sonicator (GT200: Nihonseiki Co., Tokyo, Japan) to disperse the bacterial cells trom the flocs, and plated onto the selective media and CGY medium for counts of GEMs and total heterotrophic bacteria, respectively. Plates were incubated for 1-2 days at 30"C for GEM-counts, and for 7 10 days at 25~C for total heterotrophic bacterial counts. By spraying colonies on the selective media with 0.1 mM catechol solution, pheB expression of pBH500 was also confirmed. In the experiments using the CF system, effluent samples from the settling tank were also plated and incubated in the same manner as above without sonication. On the selective media, no background colony was detected when the activated sludge samples without GEMs were plated (viable counts of the activated sludge samples on the selective media were all below 10°cfu/ml). RESULTS
Survival o f G E M s in the FD system Figure 1 shows a typical time-course of the survival of P. putida BH(pBH500), which was inoculated at 2.3 × 109cfu/ml into the F D system (SRT = 20 days) o f activated sludge. To evaluate the stability o f the plasmid in the host strains, the activated sludge samples were plated onto selective media without Sm, and the appearance o f segregants (host cells without plasmid pBH500) was estimated. The results suggested that the ratio o f segregants was 5 - 1 0 % t h r o u g h o u t the 50-day experimental period (data not shown). The phenotypes and morphologies o f several colonies formed on the selective media were investigated, and all the colonies tested showed the same phenotypes and morphologies as those o f the introduced G E M s . Therefore, the selective media used seemed to be specific to enumerate only G E M s . As shown in Fig. I, the number o f P. putida BH(pBH500) declined rapidly to 5.0 × 10 6 cfu/ml in 15 days, after which the population remained
Survival of GEMs in activated sludge relatively stable, with only a gradual decline, up to day 51 (the population size on day 51 was 3.5 x 105cfu/ml). Therefore, the survival course of the GEMs was divided into two phases--a "declining phase" and a "stable phase". This pattern occurred with all the survival courses of both P. putida BH(pBHS00) and E. coli C600(pBHS00) obtained in the experiments using the FD system under different conditions (see Figs 2 and 3); the degree of gradual decline in the stable phase seemed to be less for E. coli C600(pBHS00) than for P. putida BH(pBH500). Figure 2 shows the survival in the FD activated sludge system of GEMs inoculated at different densities. Here, the SRT was set at 20 days in all cases. The inoculum size of the introduced GEMs had little influence on rates of decline in the declining phase. However, the larger the inoculum size, at the higher level the GEMs survived in the stable phase. The effect of the SRT on the survival of introduced GEMs in the FD system was also investigated. P. putida BH(pBHS00) and E. coli C600(pBHS00) were inoculated into four flasks (FD systems) at 2.7 x 10Scfu/ml, respectively, and the SRTs of the flasks were controlled at 3, 5, l0 and 20 days as separate experiments. As shown in Fig. 3, as the SRT of the system decreased, the population size of GEMs surviving in the stable phase (cfu/ml) increased, in spite of decreases in the mixed liquor
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I I 0 20 40 Time (day) Fig. 3. Survival of E.coli C600(pBH500) (A) and P. putida BH(pBHS00) (B) in the FD activated sludge system at the SRTs of 20 (-. -), 10 ( - - - ) , 5 (" ") and 3 days (-.. -). (a), (b), (c), (d) are the theoretically expected declines due to sludge wastage corresponding to SRTs of 20, 10, 5 and 3 days, respectively. suspended solids (MLSS; MLSS in the stable phase were approx. 600-700, 1000-1100, 2000-2200 and 2300-2500mg/1 at SRTs of 3, 5, 10 and 20 days, respectively). Survival o f GEMs in the CF system
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E. coil C600(pBHS00) was introduced into the CF activated sludge system (SRT = 15 days) and monitored (Fig. 4). The GEM population showed a rapid decline from 1.6 x 106 to 7.8 x 104cfu/ml in 2 days followed by a gradual decline; a stable population of approx. 103-104cfu/ml was maintained from days 11-35. Thus, a survival course consisting of a
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Time (day) Fig. 2. Survival of E. coli C600(pBHS00) (A) and P. putida BH(pBHS00) (B) in the FD activated sludge system (SRT = 20 days) with different inoculum sizes. Cells of GEMs were recovered from 50 ( - ' - ) , 5 ( - - - ) , 0.5 ('- .) and 0.05 ml (. . . . ) of preculture were inoculated. (d) is the theoretically axpected decline due to sludge wastage corresponding to an SRT of 20 days. WR28/7--K
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Time (day) Fig. 4. Survival of E. coli C600(pBHS00) in the CF activated sludge system (SRT = 15 days). Viable counts of total heterotrophic bacteria in the aeration tank ( a ) and effluent ( + ) , and the GEM in the aeration tank (O) and effluent (A) are shown.
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Fig. 5. Survival of P. putida BH(pBH500) in the CF activated sludge system in run I(A), run 2(B), run 3(C), run 4(D) and run 5(E). Viable counts of total heterotrophic bacteria in the aeration tank (0) and effluent ( + ), and the GEM in the aeration tank (O) and effluent (A) are shown. Repeated inoculations of the GEM followed by l-h (a) or 24-h (b) feed interruptions, and addition of phenol to the wastewater (c) are indicated.
declining phase and a stable phase was observed in the CF as well as in the FD system. The survival course of P. putida BH(pBH500) in the CF system are shown in Fig. 5. Five experiments (runs 1-5) were carried out to discover the operational parameters affecting the survival of the GEM in the CF system. P. putida BH(pBH500) was inoculated at 4.4 x 106cfu/ml and the SRT was set at 15 days in run 1 [Fig. 5(A)]. The GEM population also showed a rapid decline (to 1.8 x 103cfu/ml in 10 days) followed by a stable phase (at 103 cfu/ml). In run 2, the inoculation of the GEM was repeated on days 2, 4, 6 and 20 in addition to day 0 [Fig. 5(B)]. Although the inoculum size affected the survival of the GEMs in the FD system, in the CF system repeated inoculation did not enhance the GEM survival. Besides run 1, in which the SRT was 15 days, the SRTs were set at 30 and 5 days in runs 3 and 4, respectively [Fig. 5(C) and (D)], and the effect on the
survival of the GEM in the CF system was evaluated. The population sizes of the GEM retained in the stable phase at SRTs of 5, 15 and 30 days were approx. 104, 103 and 102 cfu/ml, respectively, thus an increase in the ecological stability of the GEM with a decrease in the SRT was observed in the CF as well as in the FD system. However, repeated inoculation in runs 3 and 4 also did not raise the GEM population. Substrates, which can be used specifically by introduced GEMs or by particular indigenous bacteria, seem to affect the survival of GEMs in the activated sludge process. In run 5, phenol, which supports the growth of P. putida BH(pBH500) as a sole carbon source, was fed from the day 21 contained in the synthetic wastewater at a concentration of 100 mg/1 (the GEM was reinoculated on day 20), and its affect on the survival of the GEM was investigated [Fig. 5(E)]. As shown in the figure, no distinct change in the GEM population occurred.
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Survival of GEMs in activated sludge The GEM was detected in the effluent from the settling tank at approx. 1/100 the density of that in the aeration tank in all the experiments using the CF system with the exception of run 3, in which the GEM in the effluent was considered to be less than 10° cfu/ml, the detectable limit of the method used in this study.
DISCUSSION
All the experimental results showed that the GEMs introduced into the activated sludge could maintain their populations at relatively stable levels after the declining period. This suggests the possibility of utilizing the abilities of GEMs in the activated sludge process, although it is desirable to establish strategies to maintain them at higher levels. Accordingly, it is important to elucidate the mechanisms and/or determine the influential parameters concerning the survival of GEMs in the activated sludge process. The rapid drop in the GEM population observed in the declining phase cannot be accounted for only by sludge wastage (see Figs 2 and 3 for theoretically expected declines of GEMs due to sludge wastage). This suggests the presence of other selective pressures, which could include predation by protozoa, competition with indigenous bacterial populations and fatal environmental effects. These are the pressures of the ecosystem to wash out lives which have dominated excessively in the ecosystem. This pressure may be called the selective pressure of the ecosystem. In the declining phase, the selective pressure of the ecosystem disordered by the introduction of GEMs is considered to be high, whereas in the stable phase the introduced GEMs have become settled as members of a relatively stable ecosystem, and the selective pressure seems to be low. In this study, two host strains, E. coli C600 and P. putida BH, were used for the breeding of the GEMs. Pseudomonas has been reported as one of the dominant genera isolated from various activated sludges, whereas enteric bacteria, including E. coli, have rarely been detected (Pike and Carrington, 1972; Pipes, 1969; van Gils, 1964). Therefore, although P. putida BH(pBHS00) seemed to be more adaptable to the activated sludge process than E. coli C600(pBH500), the survival or ecological stability of the two strains did not greatly differ from each other here. Through the experiments, certain parameters affecting the survival of GEMs were determined. The GEMs survived at higher levels in the FD system than in the CF one; the inoculum size affected the survival of GEMs considerably in the FD system, but repeated inoculations had no effects in the CF system. These results suggested that the operation mode of the activated sludge process was one of the factors that influenced GEM survival. The SRT influenced the survival of the GEMs irrespective of the operation mode--as the SRT decreased, the survival level
of the GEMs increased. Hashimoto et al. (1987) reported that the diversity of bacterial flora in activated sludge cultivated with synthetic wastewater mainly containing meat extract and peptone increased with increases in the SRT of the process. The diversity of indigenous bacterial flora might influence the survival of GEMs in activated sludge. On the other hand, phenol did not act as a selective pressure with P. putida BH(pBH500). This suggests that phenol is not especially required as a substrate to accelerate the growth of the GEM in a complex ecosystem of mixed substrates/microorganisms such as in the activated sludge process. Further studies on the expression and transfer of recombinant genes, assessment of hazardous effects, etc., are needed in order that the application of GEMs to wastewater treatment processes, including activated sludge processes, can be realized.
REFERENCES
Appleyard R. K. (1954) Segregation of new lysogenic types during growth of a double lysogenic strain derived from Escherichia coli K12. Genetics 39, 440-446. Bagdasarian M., Luze R., Ruckert B., Franklin F. C. H., Bagdasarian M. M., Frey J. and Timmis K. N. (1973) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derivedvectors and host-vector system for gene cloning in Pseudomonas. Gene 16, 237-247. Chapman P. J. (1988) Constructing microbial strains for degradation of halogenated aromatic hydrocarbons. In Environmental Biotechnology: Reducing Risks from Environmental Chemicals (Edited by Omenn G. S.),
pp. 81-95. Plenum Press, New York. Fujita M., Ike M. and Hashimoto S. (1991) Feasibility of wastewater treatment using geneticallyengineered microorganisms. Wat. Res. 25, 979-984. Fujita M., Ike M. and Kamiya T. (1993) Accelerated phenol removal by amplifying the gene expression with a recombinant plasmid encoding catechol 2,3-oxygenase. Wat. Res. 27, 9-13. van Gils H. W. (1964) Bacteriology of activated sludge. T.N.O. Report No. 32, Research Institute for Public Health Engineering. Hashimoto S., Fujita M. and Ike M. (1987) Study on the change of bacterial population in activated sludge process controlled by SRT. Proc. envir. Sanit. Engng Res. 23, 251-260 (in Japanese). Kellog S. T., Chatterjee D. K. and Chakrabarty A. M. (1981) Plasmid-assisted molecular breeding: new technique for enhanced biodegradation of persistent toxic chemicals. Science 214, 1133-I 135. Kobayashi H. A. (1984) Application of genetic engineering to industrial waste/wastewater treatment. In Genetic Control of Environmental Pollutants (Edited by Omenn G. S. and Hollaender A.), pp. 47-80. Plenum Press, New York. McClure N. C., Fry J. C. and Weightman A. J. (1991a) Genetic engineering for wastewater treatment. J. I W E M 5, 608-616. McClure N. C., Fry J. C. and Weightman A. J. (1991b) Survival and catabolic activity of natural and genetically engineered bacteria in a laboratory-scale activated-sludge unit. Appl. envir. Microbiol. 57, 366-373. McClure N. C., Weightman A. J. and Fry J. C. (1989) Survival of Pseudomonas putida UWC 1 containing cloned catabolic genes in a model activated-sludge unit. Appl. envir. Microbiol. 55, 2627-2634.
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Pike E. B. and Carrington E. G. (1972) Recent development in the study of bacteria in the activated-sludge process. Wat. Pollut. Control 71, 583-600. Pike E. B., Carrington E. G. and Ashburner P. A. (1972) An evaluation of procedures for enumerating bacteria in activated sludge. J. appl. Bact. 35, 309-32l. Pipes W. O. (1969) The ecological approach to the study of activated sludge. Adv. appl. Microbiol. 8, 61 103.
Stotzky G. and Babich H. (1986) Survival of, and genetic transfer by, genetically engineered bacteria in natural environments. Adv. appl. Microbiol. 31, 93-138. Timmis K. N., Rojo F. and Ramos J. L. (1988) Prospects of laboratory engineering of bacteria to degrade pollutants. In Environmental Biotechnology: Reducing Risks from Environmental Chemicals (Edited by Omenn G. S.), pp. 61-79. Plenum Press, New York.
Pergamon
O043-1354(93)EOO29-R
War. Res. Vol. 28, No. 7, pp. 1667-1672, 1994 Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/94 $7.00+ 0.00
OPERATION PARAMETERS AFFECTING THE SURVIVAL OF GENETICALLY ENGINEERED MICROORGANISMS IN ACTIVATED SLUDGE PROCESSES MASANORIFUJITA*~, MICHIHIKOIKE and KAZUYAUESUGI Department of Environmental Engineering, Osaka University, Yamadaoka, Suita, Osaka 565, Japan (First received April 1993; accepted in revised form November 1993)
Abstract The survival of genetically engineered microorganisms (GEMs) harboring recombinant plasmid pBHS00, containing catechol 2,3-oxygenase encoding gene, in model activated sludge processes was investigated. Escherichia coil C600 (pBH500) and Pseudomonas putida BH(pBH500) were inoculated into activated sludge and cultivated in the fill and draw (FD) and continuous flow (CF) systems under different conditions. In both systems, the populations of introduced GEMs declined rapidly during the initial period (5-10 days for the FD system and 5-15 days for the CF system), after which they remained relatively stable. In the FD system, the larger the inoculum size, the higher the population level at which the GEMs remained stable. However, in the CF system, repeated inoculation did not improve the survival ofP. putida BH(pBH500). The sludge retention time (SRT) affected the survival of GEMs considerably in both systems; as the SRT of the system was decreased, the survival populations of GEMs increased. The presence of phenol, which can support the growth of P. putida BH(pBH500), did not influence its survival. Key words--survival of genetically engineered microorganisms, activated sludge process, sludge retention time, inoculum size, fill and draw system, continuous flow system, Pseudomonas putida, Escherichia coli, selective pressure of ecosystem
INTRODUCTION The potential for the improvement of wastewater treatment by using genetically engineered microorganisms (GEMs), especially in the effective removal of xenobiotic or toxic compounds, has been recognized by many researchers (Fujita et al., 1991; Kobayashi, 1984; McClure et al., 1991a). GEMs capable of degrading a wide variety of xenobiotic compounds or showing enhanced degradation activities have already been constructed (Fujita et al., 1993; Kellog et al., 1981; Chapman, 1988; Timmis et al., 1988). However, before this approach can be realized, the problem of the ecological stability of GEMs must be solved. Ecological stability refers to the length of time that the GEMs can survive and express their useful activities in the mixed microbial flora of wastewater treatment processes. If a G E M has a low ecological stability, it may soon disappear from the process and the treatment efficiency will thus be little improved, even if a G E M with a high xenobiotic compound degradation rate is constructed. The survival of introduced bacterial strains, including GEMs, in the mixed microbial flora of natural environments or microcosms, such as soils, sediments, sewage and pond/lake water has been dealt with in numerous studies (Stotsky and Babich, 198@
*Author to whom all correspondence should be addressed.
As for the survival in wastewater treatment processes, McClure et al. (1991 b, 1989) investigated the survival of natural and genetically engineered bacteria capable of degrading 3-chlorobenzoate in a laboratory-scale activated sludge unit, and pointed out the importance of choosing strains that are well-adapted to the environmental conditions for the successful use of microbial inoculation. However, to date, little is known about the mechanism of GEM survival in wastewater treatment processes. This study was designed to investigate the survival of two GEMs, Escherichia coil C600(pBHS00) and Pseudomonas putida BH(pBH500) (Fujita et al., 1991, 1993) harboring a recombinant plasmid in both fill and draw (FD) and continuous flow (CF) model activated sludge systems, which simulated fed-batch/ batch and conventional activated sludge treatment processes, respectively. An attempt was made to determine some of the operational parameters which might influence the survival of GEMs.
METHODS
MATERIALSAND Bacterial strains and plasmid E. coli C600 (Appleyard, 1954) and phenol and benzoateutilizing P. putida BH were used as host strains of recombinant plasmid pBHS00 (Fujita et al., 1991). Recombinant plasmid pBH500 was constructed by inserting the 5.65 kbDNA fragment containing catechol 2,3-oxygenase (C230), . which catalyses the conversion of catechol into 2-hydroxymuconic semiaidehyde (2-HMS), encoding gene (pheB)
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from the chromosome of P. putida BH, into a broad host range vector plasmid pKT230 (Bagdasarian et al., 1973). This plasmid gives the host strains resistance to streptomycin (Sm) and the constitutive expression of pheB. Colonies expressing pheB can be identified by spraying with 0.I mM catechol solution. Positive colonies quickly turn yellow due to the formation of 2-HMS. The phenotypes of the bacterial strains and plasmid pBH500 are listed in Table 1.
Media Both GEMs, E. coli C600(pBH500) and P. putida BH(pBH500), were routinely maintained on CGY medium (Pike et al., 1972) supplemented with Sm, and grown in modified L broth (LM broth) containing 10g of Bacto peptone, 5 g of Bacto-yeast extract and 5 g of NaC1 in I liter of deionized water (pH = 7.4) with Sm at 28~'C on a rotary shaker (120 rpm). CGY medium was also used for enumeration of total heterotrophic bacteria. Counts of the GEMs were carried out with the selective media as follows. E. coil C600(pBH500) was enumerated by using deoxycholate agar (Eiken Chemical Co., Tokyo, Japan) for isolation of enteric bacteria with 100 mg/l Sm and P. putida BH(pBH500) by benzoate medium containing l g of K~HPO4, 1 g of (NH4)2SO 4, 0.1 g of MgSO4, 0.02g of FeC13, 0.1g of NaC1, 0.1g of CaCl 2, 0.5g of sodium benzoate and 15 g of agar (pH = 7.4) in 1 liter of deionized water with 50 mg/l Sm. Model activated sludge systems The activated sludge used in this study had been acclimated to synthetic wastewater containing 0.2 g of meat extract, 0.3 g of peptone, 0.05 g of urea, 0.015 g of NaCI, 0.007 g of KC1, 0.007 g of CaC12, 0.005 g of MgSO4 and Na 2HPO4 in 1 liter of tap water for more than 2 years by the fill and draw technique. Two model activated sludge systems were constructed and operated as follows. Fill and draw (FD) system. Activated sludge treatment by the FD system was carried out by shake culture. GEMs were grown overnight, pelleted by centrifugation (10,000g, 15 min) and added to 100 ml of the activated sludge (MLSS conc. = approx. 2000 mg/l). The sludge was cultivated in a 300-ml Erlenmeyer flask at 25°C on a rotary shaker (100 rpm). After 21 25-h cultivation, a specific volume of waste sludge was withdrawn to control the sludge retention time (SRT) of the system, and the rest was settled for 30 min, The supernatant (85-90% of the total volume) was then discarded, and the sterilized double-strength synthetic wastewater above was added up to 100 ml. These operations were repeated daily. Continuous flow (CF) system. The CF system consisted of an aeration tank (working volume = 5 l) with a liquid overflow to a settling tank (working volume = 1 liter), the sludge being returned to the aeration tank (4,31/day). Double-strength synthetic wastewater was fed continuously (5 1/day) to the aeration tank. Aeration was conducted at 0.4 vvm of air with an air pump and diffuser attached at the
Table I. Bacterial strains and plasmid used in this study Strain or plasmid Phenotype* E. coli C600 thi ,leu ,thr ,Sm ~ P. putida BH Ben+, Phe+, Sm' Isolated from activated sludge pBH500
Sm~, C230 Constructed by inserting 5.65kb-DNA fragment from the chromosomeof P. putida BH into pKT230 *Abbreviations:requirementsfor thiamine(thi- ), leucine(leu ) and threonine(thr- ); growth on benzoate(Ben+ ) and phenol(Phe+ ); resistant to (Sm~)or sensitiveto (Sin') streptomycin;constitutive expression of pheB (C230).
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Fig. 1. Survival of P. putida BH(pBH500) in the FD activated sludge system (SRT = 20 days). Viable counts of total heterotrophic bacteria ( 0 ) and the GEM (©) are shown. bottom of the tank, in which the temperature was maintained at 25°C. Waste sludge was withdrawn from the aeration tank. GEMs were harvested in the same manner as described above and added to the aeration tank. The feeding ofwastewater was stopped after the inoculation for 1 or 24 h (after the first inoculation on day 0, the feeding was stopped for 24 h).
Enumeration of GEMs Activated sludge samples withdrawn were harvested by centrifugation (10,000g, 10rain) and resuspended in the same volume of 5 mg/l s,~dium tripolyphosphate solution (tpp.). A 50 ml sample diluted 10-fold with tpp. was treated with a sonicator (GT200: Nihonseiki Co., Tokyo, Japan) to disperse the bacterial cells trom the flocs, and plated onto the selective media and CGY medium for counts of GEMs and total heterotrophic bacteria, respectively. Plates were incubated for 1-2 days at 30"C for GEM-counts, and for 7 10 days at 25~C for total heterotrophic bacterial counts. By spraying colonies on the selective media with 0.1 mM catechol solution, pheB expression of pBH500 was also confirmed. In the experiments using the CF system, effluent samples from the settling tank were also plated and incubated in the same manner as above without sonication. On the selective media, no background colony was detected when the activated sludge samples without GEMs were plated (viable counts of the activated sludge samples on the selective media were all below 10°cfu/ml). RESULTS
Survival o f G E M s in the FD system Figure 1 shows a typical time-course of the survival of P. putida BH(pBH500), which was inoculated at 2.3 × 109cfu/ml into the F D system (SRT = 20 days) o f activated sludge. To evaluate the stability o f the plasmid in the host strains, the activated sludge samples were plated onto selective media without Sm, and the appearance o f segregants (host cells without plasmid pBH500) was estimated. The results suggested that the ratio o f segregants was 5 - 1 0 % t h r o u g h o u t the 50-day experimental period (data not shown). The phenotypes and morphologies o f several colonies formed on the selective media were investigated, and all the colonies tested showed the same phenotypes and morphologies as those o f the introduced G E M s . Therefore, the selective media used seemed to be specific to enumerate only G E M s . As shown in Fig. I, the number o f P. putida BH(pBH500) declined rapidly to 5.0 × 10 6 cfu/ml in 15 days, after which the population remained
Survival of GEMs in activated sludge relatively stable, with only a gradual decline, up to day 51 (the population size on day 51 was 3.5 x 105cfu/ml). Therefore, the survival course of the GEMs was divided into two phases--a "declining phase" and a "stable phase". This pattern occurred with all the survival courses of both P. putida BH(pBHS00) and E. coli C600(pBHS00) obtained in the experiments using the FD system under different conditions (see Figs 2 and 3); the degree of gradual decline in the stable phase seemed to be less for E. coli C600(pBHS00) than for P. putida BH(pBH500). Figure 2 shows the survival in the FD activated sludge system of GEMs inoculated at different densities. Here, the SRT was set at 20 days in all cases. The inoculum size of the introduced GEMs had little influence on rates of decline in the declining phase. However, the larger the inoculum size, at the higher level the GEMs survived in the stable phase. The effect of the SRT on the survival of introduced GEMs in the FD system was also investigated. P. putida BH(pBHS00) and E. coli C600(pBHS00) were inoculated into four flasks (FD systems) at 2.7 x 10Scfu/ml, respectively, and the SRTs of the flasks were controlled at 3, 5, l0 and 20 days as separate experiments. As shown in Fig. 3, as the SRT of the system decreased, the population size of GEMs surviving in the stable phase (cfu/ml) increased, in spite of decreases in the mixed liquor
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E. coil C600(pBHS00) was introduced into the CF activated sludge system (SRT = 15 days) and monitored (Fig. 4). The GEM population showed a rapid decline from 1.6 x 106 to 7.8 x 104cfu/ml in 2 days followed by a gradual decline; a stable population of approx. 103-104cfu/ml was maintained from days 11-35. Thus, a survival course consisting of a
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Fig. 5. Survival of P. putida BH(pBH500) in the CF activated sludge system in run I(A), run 2(B), run 3(C), run 4(D) and run 5(E). Viable counts of total heterotrophic bacteria in the aeration tank (0) and effluent ( + ), and the GEM in the aeration tank (O) and effluent (A) are shown. Repeated inoculations of the GEM followed by l-h (a) or 24-h (b) feed interruptions, and addition of phenol to the wastewater (c) are indicated.
declining phase and a stable phase was observed in the CF as well as in the FD system. The survival course of P. putida BH(pBH500) in the CF system are shown in Fig. 5. Five experiments (runs 1-5) were carried out to discover the operational parameters affecting the survival of the GEM in the CF system. P. putida BH(pBH500) was inoculated at 4.4 x 106cfu/ml and the SRT was set at 15 days in run 1 [Fig. 5(A)]. The GEM population also showed a rapid decline (to 1.8 x 103cfu/ml in 10 days) followed by a stable phase (at 103 cfu/ml). In run 2, the inoculation of the GEM was repeated on days 2, 4, 6 and 20 in addition to day 0 [Fig. 5(B)]. Although the inoculum size affected the survival of the GEMs in the FD system, in the CF system repeated inoculation did not enhance the GEM survival. Besides run 1, in which the SRT was 15 days, the SRTs were set at 30 and 5 days in runs 3 and 4, respectively [Fig. 5(C) and (D)], and the effect on the
survival of the GEM in the CF system was evaluated. The population sizes of the GEM retained in the stable phase at SRTs of 5, 15 and 30 days were approx. 104, 103 and 102 cfu/ml, respectively, thus an increase in the ecological stability of the GEM with a decrease in the SRT was observed in the CF as well as in the FD system. However, repeated inoculation in runs 3 and 4 also did not raise the GEM population. Substrates, which can be used specifically by introduced GEMs or by particular indigenous bacteria, seem to affect the survival of GEMs in the activated sludge process. In run 5, phenol, which supports the growth of P. putida BH(pBH500) as a sole carbon source, was fed from the day 21 contained in the synthetic wastewater at a concentration of 100 mg/1 (the GEM was reinoculated on day 20), and its affect on the survival of the GEM was investigated [Fig. 5(E)]. As shown in the figure, no distinct change in the GEM population occurred.
1671
Survival of GEMs in activated sludge The GEM was detected in the effluent from the settling tank at approx. 1/100 the density of that in the aeration tank in all the experiments using the CF system with the exception of run 3, in which the GEM in the effluent was considered to be less than 10° cfu/ml, the detectable limit of the method used in this study.
DISCUSSION
All the experimental results showed that the GEMs introduced into the activated sludge could maintain their populations at relatively stable levels after the declining period. This suggests the possibility of utilizing the abilities of GEMs in the activated sludge process, although it is desirable to establish strategies to maintain them at higher levels. Accordingly, it is important to elucidate the mechanisms and/or determine the influential parameters concerning the survival of GEMs in the activated sludge process. The rapid drop in the GEM population observed in the declining phase cannot be accounted for only by sludge wastage (see Figs 2 and 3 for theoretically expected declines of GEMs due to sludge wastage). This suggests the presence of other selective pressures, which could include predation by protozoa, competition with indigenous bacterial populations and fatal environmental effects. These are the pressures of the ecosystem to wash out lives which have dominated excessively in the ecosystem. This pressure may be called the selective pressure of the ecosystem. In the declining phase, the selective pressure of the ecosystem disordered by the introduction of GEMs is considered to be high, whereas in the stable phase the introduced GEMs have become settled as members of a relatively stable ecosystem, and the selective pressure seems to be low. In this study, two host strains, E. coli C600 and P. putida BH, were used for the breeding of the GEMs. Pseudomonas has been reported as one of the dominant genera isolated from various activated sludges, whereas enteric bacteria, including E. coli, have rarely been detected (Pike and Carrington, 1972; Pipes, 1969; van Gils, 1964). Therefore, although P. putida BH(pBHS00) seemed to be more adaptable to the activated sludge process than E. coli C600(pBH500), the survival or ecological stability of the two strains did not greatly differ from each other here. Through the experiments, certain parameters affecting the survival of GEMs were determined. The GEMs survived at higher levels in the FD system than in the CF one; the inoculum size affected the survival of GEMs considerably in the FD system, but repeated inoculations had no effects in the CF system. These results suggested that the operation mode of the activated sludge process was one of the factors that influenced GEM survival. The SRT influenced the survival of the GEMs irrespective of the operation mode--as the SRT decreased, the survival level
of the GEMs increased. Hashimoto et al. (1987) reported that the diversity of bacterial flora in activated sludge cultivated with synthetic wastewater mainly containing meat extract and peptone increased with increases in the SRT of the process. The diversity of indigenous bacterial flora might influence the survival of GEMs in activated sludge. On the other hand, phenol did not act as a selective pressure with P. putida BH(pBH500). This suggests that phenol is not especially required as a substrate to accelerate the growth of the GEM in a complex ecosystem of mixed substrates/microorganisms such as in the activated sludge process. Further studies on the expression and transfer of recombinant genes, assessment of hazardous effects, etc., are needed in order that the application of GEMs to wastewater treatment processes, including activated sludge processes, can be realized.
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Stotzky G. and Babich H. (1986) Survival of, and genetic transfer by, genetically engineered bacteria in natural environments. Adv. appl. Microbiol. 31, 93-138. Timmis K. N., Rojo F. and Ramos J. L. (1988) Prospects of laboratory engineering of bacteria to degrade pollutants. In Environmental Biotechnology: Reducing Risks from Environmental Chemicals (Edited by Omenn G. S.), pp. 61-79. Plenum Press, New York.