The Incorporation of Farnesol-2-14C into Ipomeamarone

The survival of genetically engineered and wild-type Pseudomonas putida PpY101, that contained a recombinant plasmid pSR134 conferring mercury resistance, were monitored in andosol and sand microcosms. The survival of genetically engineered and wild-type P. putida was not significantly different in andosol. The population change of the two strains was dissimilar in andosol and sand. The survival of genetically engineered and wild-type P. putida strains was affected by the water content of andosol, and increased with the increment of the water content. The impact of the addition of genetically engineered and wild-type P. putida strains on indigenous bacteria and fungi was examined. Inoculation of both strains had no apparent effect on the density of indigenous microorganisms.     

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria

The survival of genetically engineered Erwinia carotovora L-864, with a kanamycin resistance gene inserted in its chromosome, was monitored in the water and sediment of aquatic microcosms. The density of genetically engineered and wild-type E. carotovora strains declined at the same rate, falling in 32 days below the level of detection by viable counts. We examined the impact of the addition of genetically engineered and wild-type strains on indigenous bacteria belonging to specific functional groups important in nutrient cycling. For up to 16 days, the densities of total and proteolytic bacteria were significantly higher (P < 0.05) in microcosms inoculated with genetically engineered or wild-type E. carotovora, but by 32 days after inoculation, they had decreased to densities similar to those in control microcosms. Inoculation of genetically engineered or wild-type E. carotovora had no apparent effect on the density of amylolytic and pectolytic bacteria in water and sediment. Genetically engineered and wild-type E. carotovora did not have significantly different effects on the densities of specific functional groups of indigenous bacteria (P > 0.05).     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments.

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

Monitoring of Biodegradative Pseudomonas putida Strains in Aquatic Environments Using Molecular Techniques

Abstract Monitoring strategies were developed to track non–genetically engineered Pseudomonas putida strains in the open environment. The strain E1 was used for four years for the biodegradation of phenolic compounds in industrial wastewater in P?lva, Estonia. In this study we used the strain E2 which is a non-carbenicillin-resistant variant of the strain E1. Both strains have a deletion of approximately 34 kb in the TOL plasmid pWW0 which served as a basis for discrimination from indigenous bacteria by molecular analyses. Other targets used for PCR and DNA hybridization were the xylE gene and a sequence located in the left-handed region of to the transposon Tn4652. In laboratory tests we demonstrated that two cells inoculated into 20 ml of river water could be detected against a background of more than 10⁷ colony forming units (CFUs) by a combination of growth on selective media and molecular analysis. Using the same combination of methods in a deliberate release experiment, detection of the released strain was possible only to 32 h after release. It is assumed that the released strains did not survive in the aquatic ecosystem, mainly due to the high dilution rate. The combination of cultivation on selective media and molecular analyses proved useful for tracking Pseudomonas putida strain E2 in an aquatic environment. Received: 29 March 1996; Accepted: 1 April 1996     

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.Correspondence to: M.G. Höfle     

Comparison of in-situ biodegrading abilities of Pseudomonas putida mutants: leuB− auxotroph,fliC− non-motility,and cheA− non-chemotaxis

Bioremediation of pollutants in natural environments is affected by many factors, such as bacterial survival, motility, and chemotaxis. However, these roles in in-situ biodegradation of organophosphorus pesticides have not been examined extensively. In this paper, a highly effective methyl-parathion (MP) degrading strain, Pseudomonas putida DLL-1, which also demonstrates motile ability and chemotactic response toward MP, was selected as the research material. A leuB^? auxotroph mutant A3-27 and fliC^? non-motility mutant a4-8 were first constructed by random insertion of the kanamycin gene into the chromosome of P. putida DLL-1 with the mini-transposon system. Biodegradation of MP in liquid medium and soil microcosms by A3-27, a4-8 and a previously constructed cheA^? non-chemotaxis mutant P. putida DAK were compared. The kinetic parameters for MP degradation were all similar in the well-mixed liquid systems. However, in soil microcosms, all the three mutants had lower degrading rates compared with wild-type P. putida DLL-1. The auxotroph mutant A3-27 had the lowest degrading rate and could only degrade 25.7–34.2% MP in 5 days, and the non-motility mutant a4-8 and non-chemotaxis mutant DAK could only degrade 53.5–68.1% and 64.3–85.7% MP, respectively. This paper emphasizes, for the first time, the use of non-auxotroph bacteria for efficient removal of organophosphorus pesticides in contaminated sites, and also points out the importance of select microorganisms with specific motile or chemotactic affinities in optimizing pesticide bioremediation.     

Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.     

Persistence of genetically engineeredErwinia carotovora in perturbed and unperturbed aquatic microcosms and effect on recovery of indigenous bacteria

Genetically engineeredErwinia carotovora persisted significantly longer in thermally perturbed microcosms (35 days) than in nonstressed microcosms (5 days). Decreased pressure of competitors and predators and increased nutrient availability were examined as the most probable reasons for greater vulnerability of perturbed microcosms to colonization by genetically engineered microorganisms (GEMs). Indigenous bacteria that competed with GEMs for the same nutrient sources (protein, cellulose, pectate) were present immediately after perturbation in densities one to two orders of magnitude lower than in unperturbed microcosms, but their populations increased to densities significantly higher than in unperturbed microscosms 10 to 15 days after inoculation. Predators of bacteria (protozoans, cladocerans, nematodes, and rotifers) were present during the experiment in unperturbed microcosms, while dense populations of bacteriovorous nanoflagellates developed in perturbed microcosms. Preemptive inoculation of perturbed microcosms with GEMs did not have a longlasting effect on the recovery of total, proteolytic, cellulolytic, and pectolytic bacteria in perturbed microscosms, indicating the absence of competitive exclusion.     

Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.     

Efficacy in aquatic microcosms of a genetically engineered pseudomonad applicable for bioremediation

A genetically engineered microorganism (GEM), Pseudomonas sp. B13 FRI (pFRC20P) (abbreviated FR120), has previously been engineered to simultaneously mineralize mixtures of methylated and chlorinated benzoic acids and phenols through a modified ortho cleavage pathway. In this study, its performance was investigated both in different types of aquatic microcosms and in pure culture to determine (1) if under simulated in situ conditions the genetically engineered pathway effectively removes mixtures of model pollutants simultaneously, quickly, and completely; (2) where the optimum pollutant concentration range for this activity lies; and (3) how physical, chemical, and biological factors in the microcosms influence degradation rates. Growth and degradation parameters of FR 120 in pure culture were determined with 3-chlorobenzoate (3CB), 4-methylbenzoate (4MB), and equimolar mixtures of both as carbon sources. These substrates were degraded simultaneously, albeit with different degradation velocities, by FR120. The optimum growth concentrations for 3CB and 4MB were 3.0 mm and 2.1 mm, respectively, and the inhibition constants (K_i) were 11 mm (3CB) and 6 mm (4MB). The pathway was induced at low concentrations of substrate (> 1 [m). The first order degradation constants (k_l) were determined with respect to substrate concentration, cell density, and temperature. In aquatic microcosms inoculated with FR120, first order degradation constants and half lives of target chemicals were calculated based on the total amount of aromatics recovered. Half lives ranged from 1.3 days to 3.0 days, depending on the target chemical and the type of microcosm. Degradation constants determined in pure culture were extrapolated to the densities of FR120, substrate concentrations, and temperature occurring in the microcosm experiments, and used to calculate theoretical half lives. In water microcosms, theoretical and observed half lives corresponded well, indicating that FR120 functioned optimally in this environment. In whole core sediment microcosms, and especially at low cell densities, the observed degradation activity was in some cases considerably higher than expected from pure culture degradation rates. This suggests that environmental conditions in the sediment were more favorable to the degradation of substituted aromatics than those in pure culture. The physiological characteristics of FR120 and its performance in aquatic microcosms make it a good candidate for bioremediation at sites contamninated with mixtures of chlorinated and methylated aromatics. Correspondence to: I. Wagner-Döbler     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Bacterial community dynamics during biostimulation and bioaugmentation experiments aiming at chlorobenzene degradation in groundwater

A set of microcosm experiments was performed to assess different bioremediation strategies, i.e., biostimulation and bioaugmentation, for groundwater contaminated with chlorobenzenes. The biodegradative potential was stimulated either by the supply of electron acceptors (air, (NO ₃ ^– ), to increase the activity of the indigenous bacterial community, or by the addition of aerobic chlorobenzene-degrading bacteria (Pseudomonas putida GJ31, Pseudomonas aeruginosa RHO1, Pseudomonas putida F1CC). Experiments were performed with natural groundwater of the aquifer of Bitterfeld, which had been contaminated with 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB), and chlorobenzene (CB). The microcosms consisted of airtight glass bottles with 800 mL of natural groundwater and were incubated under in situ temperature (13°C). Behavior of the introduced strains within the indigenous bacterial community was monitored by fluorescent in situ hybridization (FISH) with species-specific oligonucleotides. Dynamics of the indigenous community and the introduced strains within the microcosms were followed by single-strand conformation polymorphism (SSCP) analysis of 16S rDNA amplicons obtained from total DNA of the microbial community. An indigenous biodegradation potential under aerobic as well as anaerobic denitrifying conditions was observed accompanied by fast and specific changes in the natural bacterial community composition. Augmentation with P. aeruginosa RHO1 did not enhance bio-degradation. In contrast, both P. putida GJ31 as well as P. putida F1CC were capable of growing in groundwater, even in the presence of the natural microbial community, and thereby stimulating chlorobenzene depletion. P. putida GJ31 disappeared when the xenobiotics were depleted and P. putida F1CC persisted even in the absence of CB. Detailed statistical analyses revealed that community dynamics of the groundwater microbiota were highly reproducible but specific to the introduced strain, its inoculum size, and the imposed physicochemical conditions. These findings could contribute to the design of better in situ bioremediation strategies for contaminated groundwater.     

A microcosm for measuring survival and conjugation of genetically engineered bacteria in rhizosphere environments

A microcosm is described to evaluate and measure bacterial conjugation in the rhizosphere of barley and radish with strains ofPseudomonas cepacia. The purpose was to describe a standard method useful for evaluating the propensity of genetically engineered microorganisms (GEMs) to transfer DNA to recipient bacteria. Results demonstrated the formation of transconjugants from the rhizosphere of each plant 24 h after inoculation. Transconjugant populations peaked at 1.8 × 10² colony forming units (CFU)/g root and associated soil in barley and 2.0×10² CFU/g root and associated soil in radish; they then declined over the next five days of the experiment. No significant differences were found in the survival of transconjugant populations monitored from the two plant species. The microcosm was also used to document the formation of false positive transconjugants, which resulted from donor and recipientP. cepacia mating on the surface of selective agar plates instead of in microcosms. Transconjugants resulting from such plate mating occurred in substantial numbers during the first 5 days of the experiment but declined to undetectable numbers by day 7. The use of nalidixic acid was investigated to determine the magnitude of plate mating. The number of transconjugants detected from radish rhizosphere was reduced by two orders of magnitude by including nalidixic acid in the plating medium; this indicated that 99% of the transconjugants were a result of plate mating.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Mercury bioremediation by engineered Pseudomonas putida KT2440 with adaptationally optimized biosecurity circuit

Hazardous materials, such as heavy metals, are the major sources of health risk. Using genetically modified organisms (GMOs) to dispose heavy metals has the advantages of strong environmental compatibility and high efficiency. However, the biosecurity of GMOs used in the environment is a major concern. In this study, a self-controlled genetic circuit was designed and carefully fine-tuned for programmable expression in Pseudomonas putida KT2440, which is a widely used strain for environmental bioremediation. The cell behaviours were controlled by automatically sensing the variation of Hg²⁺ concentration without any inducer requirement or manual interventions. More than 98% Hg²⁺ was adsorbed by the engineered strain with a high cell recovery rate of 96% from waterbody. The remaining cells were killed by the suicide module after the mission was accomplished. The escape frequency of the engineered P. putida strain was lower than 10⁻⁹, which meets the recommendation of US NIH guideline for GMOs release (<10⁻⁸). The same performance was achieved in a model experiment by using natural lake water with addition of Hg²⁺. The microbial diversity analysis further confirmed that the remediation process made little impact on the indigenous ecosystem. Thus, this study provides a practical method for environmental remediation by using GMOs.     

Monitoring genetically engineered microorganisms in freshwater microcosms

Summary The effectiveness of gene probe methods for tracking genetically engineered microorganisms (GEMs) in the environment was tested by inoculating nutrient-supplemented freshwater microcosms withAlcaligenes A5 (a naturally occurring 4-chlorobiphenyl degrader) orPseudomonas cepacia AC1100 (a genetically engineered 2, 4, 5 T-degrader) and following the fates of the introduced bacterial populations. Colony hybridization of the viable heterotrophic bacterial populations and dot blot hybridization of DNA recovered from the total microcosm microbial communities showed persistence of bothAlcaligenes A5 andP. cepacia AC1100 in the microcosms in the presence and absence of the xenobiotic substrates that these organisms biodegrade. Although there was a gradual decline in the added populations, both of the bacterial populatins were still detected in the microcosms two months after their introduction into the microcosms. Addition of 2, 4, 5-T enhanced the survival ofP. cepacia AC1100 — and 4-chlorobiphenyl addition resulted in increased levels ofAlcaligenes A5. The results indicate that both organisms may persist for very long periods in freshwater habitats.     

Microbial trophic interactions in aquatic microcosms designed for testing genetically engineered microorganisms: A field comparison

Microcosms may potentially be used as tools for evaluating the fate and effects of genetically engineered microorganisms released into the environment. Extrapolation of data to the field, however, requires that the correspondence between microcosm and field is known. Microbial trophic interactions within the microbial loop were compared quantitatively and qualitatively between field and microcosms containing estuarine water with and without intact sediment cores. The comparison showed that whereas proportions between trophic levels in microcosms were qualitatively similar to those in the field, rates of microbial processes were from 25 to 40% lower in microcosms. Nitrogen cycling was disrupted in microcosms incubated in the dark to eliminate primary production. Examination of the microbial parameters further suggests that sediment in microcosms may be an important factor regulating the bacterial trophic level. These results demonstrate that analysis of microbial trophic interactions is a sensitive method for the field comparison of aquatic microcosms and a potentially useful tool in the risk assessment of genetically engineered microorganisms. Offprint requests to: N. Kroer.     

Recombinant and wild-type Pseudomonas aureofaciens strains introduced into soil microcosms: effect on decomposition of cellulose and straw

The effect of a genetically engineered Pseudomonas aureofaciens (Ps3732RNL11) strain (GEM) and the parental wild-type (Ps3732RN) on decomposition of cellulose paper, straw and calico cloth was assessed after 18 weeks incubation in laboratory soil microcosms. Effect(s) of inoculum density (10³, 10⁵, and 10⁸ cells/ g dry soil) and single versus multiple bacterial inoculations were also investigated. Cellulose paper was completely decomposed after 18 weeks in all treatments. There were no significant differences (95% level), between treatments, in percentage decomposition of either straw or calico cloth. Recovery of the GEM at 18 weeks, using viable plating, was limited to treatments originally receiving 10⁸ cells/g dry soil. Log 1.8 CFU/g dry soil were recovered from the single dose treatment while log 4.2 CFU/g dry soil were recovered from the multiple dose treatment Biolog metabolic tests were used to determine if the GEM or parental wild-type had any effect on overall carbon utilization in soil. Results suggested they did not. Detection of the recombinant lacZY gene sequence in soil using PCR suggested the possibility of viable but nonculturable cells and/or persistence of chromosomal DNA.