Search

Tremendous advances have recently been made in the development of molecular tools for analysis of microbial populations in the environment. However, an appropriate scientific basis for quantification of new molecular data must exist to effectively use these tools toward increased understanding of complex waste treatment environments and implementation of corrective actions to maintain or improve system performance. In particular, molecular tools are gaining widespread use in the study of activated-sludge microbial communities and have the potential to improve monitoring and control of wastewater treatment processes. The authors have created a Web-accessible database, the Activated Sludge Biomolecular Database, which provides a scientific basis for interpreting activated-sludge biomolecular information. The database achieves its goal by accumulating and disseminating a large body of knowledge relating the presence and quantity of specific biomolecules to process design, operating conditions, and wastewater characteristics.

The effect of solids retention time (SRT) on ammonia and nitrite-oxidizing bacteria was measured by Nitrosomonas oligotropha-like ammonia monooxygenaseA and Nitrospira16S rDNA competitive polymerase chain reaction assays in a complete-mix, bench-scale, activated-sludge system. During steady-state operation, nitrification was complete in the 20- and 10-day SRT reactors, nearly complete in the 5-day SRT reactor, and incomplete in the 2-day SRT reactor (76% ammonia oxidation and 85% nitrite oxidation). Total microbes, measured by dot-blot hybridizations, ranged from 3 × 1011to 3 × 1012cells/L, and increased with increasing SRTs. The concentration of the ammonia-oxidizer N. oligotrophadropped 100-fold from the 20-day SRT (5 × 109cells/L) to the 2-day SRT (=4 × 107cells/L). Thus, N. oligotrophabecame a much smaller fraction of the total biomass in the poorly performing 2-day SRT reactor. The concentration of Nitrospiracells also decreased (10-fold) as the SRT was reduced from 20 days to 2 days. However, the number of Nitrospiracells was always greater than the number of N. oligotrophacells measured in each reactor (10- to 60-fold). While Nitrospiracomprised 1 to 2% of the biomass, N. oligotropharepresented only 0.04 to 0.27% of the total population. This low percentage suggests that N. oligotrophawas not a dominant ammonia oxidizer in the bench-scale systems.

To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser-induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size-selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for ~10 min and then cooled to ~25 °C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser-induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double- versus single-stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of Cot (Co = DNA concentration and t = reassociation time) versus % recovery of double-stranded DNA signal are generated for ΦX 174 Hae ΙΙΙ digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE-LIF method, more information about the samples is available than with less specific and time-consuming traditional methods of investigating DNA reassociation.

Abstract: A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 1014 bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.

Summary The microbial populations in PCB-contaminated electric power substation capacitor bank soil (TVA soil) and from another PCB-contaminated site (New England soil) were compared to determine their potential to degrade PCB. Known biphenyl operon genes were used as gene probes in colony hybridizations and in dot blots of DNA extracted from the soil to monitor the presence of PCB-degrading organisms in the soils. The microbial populations in the two soils differed in that the population in New England soil was enriched by the addition of 1000 p.p.m. 2-chlorobiphenyl (2-CB) whereas the population in the TVA capacitor bank soil was not affected. PCB degradative activity in the New England soil was indicated by a 50% PCB disappearance (gas chromatography), accumulation of chlorobenzoates (HPLC), and14CO2 evolution from14C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to thebph gene probes, their ability to produce the yellowmeta-cleavage product from 2,3-dihydroxybiphenyl (2,3-DHB), and the degradation of specific PCB congeners by individual isolates in resting cell assays. Although the TVA capacitor bank soil lacked effective PCB-degrading populations, addition of a PCB-degrading organism and 10 000 p.p.m. biphenyl resulted in a >50% reduction of PCB levels. Molecular characterization of soil microbial populations in laboratory scale treatments is expected to be valuable in the design of process monitoring and performance verification approaches for full scale bioremediation.

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.

Summary Bacteria isolated from groundwater aquifer core materials of pristine aquifers at Lula and Pickett, OK, and from a site with a history of aromatic hydrocarbon contamination and natural renovation located at Conroe, TX, were screened for the presence of plasmid DNA by alkaline or enzyme lysis and agarose gel techniques. Some of the isolates were also subjected to taxonomic tests in addition to screening for resistance to antibiotics, tolerance to heavy metal salts, and bacteriocin production. There was no significant difference in the distribution of the traits usually associated with plasmid occurrence in isolates from the three sites. These traits, which occurred at low frequencies, were not restricted to plasmid-bearing strains of the communities. Plasmids were found in isolates from all three sites, but on the average there was a significantly higher percentage of isolates containing plasmids in the samples from Conroe (19.4%) than from either Lula (1.8%) or Pickett (7.7%). The sizes of the plasmids found ranged between 3.5 and 202 kilobases but, for the Conroe samples, many more isolates (67%) contained smaller plasmids (<10 kb) rather than larger ones. No plasmids were found in bacteria recovered from naturally renovated aquifer material at the Conroe site.

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays.

The reporter bacterium, Pseudomonas fluorescens HK44 (HK44), was characterized in an immobilized state to investigate utility for deployment as a remote sensor in the subsurface. A packed-bed reactor with alginate-immobilized HK44 simulated hydrodynamic conditions such as might be found in a subsurface environment. The reporter bacterium, HK44, harbors a reporter plasmid, pUTK21, which contains a transcriptional fusion between the nahG gene in the lower pathway of the catabolic plasmic NAH7 and a luxCDABE gene cassette. The upper nah pathway and the lux pathway in pUTK21 are induced by salicylate. The lux enzymes catalyze the light reaction. HK44 demonstrated a quantitative relationship between salicylate concentration and degradation. Light intensity mimicked salicylate concentration, whereas degradation was first order in biomass and first order in salicylate concentration, with a degradation constant of 2.23 × 10⁻² dm³ g⁻¹ min⁻¹. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 491–502, 1997.

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.

Total viable aerobic, heterotrophic bacteria, total coliforms, fecal coliforms, and fecal streptococci were enumerated in samples collected at five stations located in the Upper Chesapeake Bay, December 1973 through December 1974. Significant levels of pollution indicator organisms were detected at all of the stations sampled. Highest counts were observed in samples collected at the confluence of the Susquehanna River and the Chesapeake Bay. The indicator organisms examined were observed to be quantitatively distributed independently of temperature and salinity. Counts were not found to be correlated with concentration of suspended sediment. However, significant proportions of both the total viable bacteria (53%) and fecal indicator organisms (>80%) were directly associated with suspended sediments. Correlation coefficients (r) for the indicator organisms examined in this study ranged from r= 0.80 to r= 0.99 for bottom water and suspended sediment, respectively. Prolonged survival of fecal streptococci in most of the sediment samples was observed, with concomitant reduction of the correlation coefficient from r= 0.99, fecal streptococci to total coliforms in water, to r= 0.01, fecal streptococci to fecal coliforms in sediments. The results of this study compared favorably with fecal coliforms: fecal streptococci ratios for the various sample types. Characterization of organisms beyond the confirmed most-probable-number procedure provided good correlation between bacterial indicator groups.

An investigation of mercury-resistant bacteria was undertaken to determine their role in the accumulation of mercury in a simplified food chain. Oysters (Crassostrea virginica) were maintained in a closed system, sealed aquarium with stirred, aerated water containing 10 μg of 203HgCl2per liter. Uptake of 203Hg by oysters held under control conditions was compared with that of 203Hg uptake by oysters under similar conditions except that mercury-accumulating and mercury-metabolizing species of Pseudomonas, isolated from Chesapeake Bay, were added to the experimental oysters. After incubation for 4 days, the major portion of the 203Hg in the water column was found to be associated with the microparticulate fraction, corresponding to a rise in total viable count. Mercury accumulation in the oysters was significantly higher in the gill and visceral tissue than other tissues. Mercury concentrations were 200 times greater in tissue fractions of oysters dosed with mercury-metabolizing bacteria compared with the oysters held under control conditions without mercury-metabolizing bacteria.

Abstract: Pseudomonas fluorescens HK44 is a bioluminescent bioreporter synthesizing light in the presence of naphthalene or salicylate. Upon immobilization, HK44 is useful as an in situ or on-line biosensor of bioavailable naphthalene and salicylate in waste streams or contaminated fields. The bioreporting efficacy of alginate/SrCl2-immobilized HK44 was investigated in simulated groundwater with different pH regimes. When induced with complex (salicylate plus auxiliary energy supplements) and simple (salicylate as the sole energy supplement) inducer solutions, the specific light response was steadier at pH 6 than at pH 7 in a 35-day study. There was no bioluminescence response from cells incubated in groundwater samples with pH below 6. The rate of the luminescence reaction was stable at pH 6 irrespective of the type of inducer solution, indicating the robust physiological status of the bioreporter bacteria. In addition, the quantity of light synthesized was at least one order of magnitude higher with complex inducer solution than with simple inducer solution. The numbers of viable and cultivable cells remained constant in groundwater at pH 6 and 7 (approx. 107 g−1 beads). The numbers decreased by four orders of magnitude (107 to 103) to zero in groundwaters with pH below 6. This study suggested that HK44 is useful for long-term biosensor applications in moderately acidic to neutral groundwater conditions.

We have demonstrated the efficacy of a light-generating genetic construction in describing the induction of a nah operon for the catabolism of naphthalene. A fragment from plasmid NAH7, which contains the promoter for the upper pathway of degradation, was transcriptionally fused to the lux genes of Vibrio fischeri. A Pseudomonas strain containing this construction is inducible to high levels of light production in the presence of a suitable substrate and the nahR regulatory gene product. This system was used to examine catabolic activity in a unique manner under a variety of growth conditions. Induction of bioluminescence was demonstrated to coincide with naphthalene degradation in all cases through the use of mineralization assays. A significant delay in bioluminescence and biodegradation was observed when naphthalene was added to batch cultures that were growing exponentially. These results suggest that the metabolism of naphthalene by this Pseudomonas strain is optimal when the growth rate of the culture is slow and is greatly reduced during exponential growth.

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB.

Potential bacterial biodegradation products of polychlorobiphenyls (PCBs) were evaluated as environmental mutagens.p-Chlorophenylglyoxylic acid,p-chloromandelic acid and three monochlorinated benzoic acids were not mutagenic in either the sister chromatid exchange (SCE) orSalmonella typhimurium histidine reversion mutagenesis assays. Bacterial degradation may, therefore, tend to decrease the mutagenic potential of lesser chlorinated PCBs.

A broad-host range algD-lux bioluminescent reporter plasmid was developed to examine the role of exopolymer production in biofilm function. The algD-lux reporter plasmid will allow rapid on-line in situ detection of environmental factors that induce alginate biosynthesis. The algD promoter was stimulated by factors previously reported to induce alginate production, including ethanol and NaCl, and differences were observed with different nitrogen sources. With growth on minimal media with either glucose or succinate as a carbon source, succinate had a greater inductive effect on the algD promoter. An increase in light output of 1.3-fold and 1.7-fold was seen with cultures amended with 50 and 150 mM NaCI, respectively, compared to cultures with succinate alone. NaCl induction of the algD promoter was confirmed by algD RNA slot blots. Light output increased 2.0-fold and 1.7-fold with 0.25% and 0.5% ethanol, respectively, compared with controls grown with succinate only. While the rate of algD promoter response was initially similar when either NH4 or NO3 was used as a nitrogen source, NH4-grown cultures maintained a higher light output during late log phase compared to NO3-grown cultures.

In the course of a study of the microbial degradation of polychlorinated biphenyls (PCB), 16 cultures, representing 5 genera of bacteria, were isolated. APseudomonas sp. was found to degrade mixtures of PCB (Aroclor 1254) and pure isomers, i.e., hexachlorobiphenyl (HCB). Degradation was found to be variable, depending on length of incubation, purity, and degree of chlorination of the biphenyl molecule. In addition to apparent degradation of PCB, it was observed that PCB stimulated growth and oxygen uptake. Bioaccumulation of PCB residues was suggested since14-Clabeled material derived from14C-PCB accumulated on or in the bacterial cells. The potential degradation and utilization of PCB by microorganisms suggests that this may be an important mechanism for removal or mobilization of PCB into food chains in the estuarine environment.