The activated sludge biomolecular database.

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.

A toxicity testing protocol using a bioluminescent reporter bacterium from activated sludge.

A protocol for production, storage, and use of Shock 1 (Shk1) bioreporter cells for toxicity monitoring in wastewater treatment facilities was developed. Shk1 is a bioluminescent toxicity bioreporter for activated sludge previously constructed by the incorporation of lux genes into an activated sludge microorganism.A number of factors affecting Shk1 growth and bioluminescence were examined including the growth medium, tetracycline concentration, storage conditions, and test media. Based on the results of these experiments, a toxicity testing protocol was developed that involved growth of cultures in nutrient broth with tetracycline, storage of cultures at 4 degrees C, cell activation by reinoculation into nutrient broth, and toxicity testing by cell injection into the test media. Effective use of this approach required standardized time intervals for cell growth, storage, activation and exposure in the test media. Bioluminescence from Shk1 cells was measured in nutrient broth and influent wastewater and activated sludge mixed liquor from a municipal wastewater treatment plant. Using the Shk1 toxicity testing protocol, Zn EC(50) values for bioluminescence in nutrient broth, influent wastewater, and activated sludge mixed liquor were approximately 42, 7, and 32 mg/l, respectively. Zn concentrations as low as 1 mg/l could be detected in influent wastewater. The detection limit in influent wastewater is below the Zn concentrations typically reported to affect the activated sludge process.

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis.

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors.

Degradation of polychlorinated biphenyls (PCBs) in the environment is limited by their aqueous solubility and the degradative competence of indigenous populations. Field application vectors (FAVs) have been developed in which surfactants are used to both increase the solubility of the PCBs and support the growth of surfactant-degrading strains engineered for PCB degradation. Surfactant and PCB degradation by two recombinant strains were investigated. Pseudomonas putida IPL5 utilizes both alkylethoxylate [polyoxyethylene 10 lauryl ether (POL)] and alkylphenolethoxylate [Igepal CO-720 (IGP)] surfactants as growth substrates, but only degrades the ethoxylate moiety. The resulting degradation products from the alkyl- and alkylphenolethoxylate surfactants were 2-(dodecyloxy)ethanol and nonylphenoldiethoxylates, respectively. Ralstonia eutropha B30P4 grows on alkylethoxylate surfactants without the appearance of solvent-extractable degradation products. It also degrades the 2-(dodecyloxy)ethanol produced by strain IPL5 from the alkylethoxylate surfactants. The extent of degradation of the alkylethoxylate surfactant (POL) was greater for strain IPL5 (90%) than for B30P4 (60%) as determined by the cobaltothiocyanate active substances method (CTAS). The recombinant strain B30P4::TnPCB grew on biphenyl. In contrast, the recombinant strain IPL5::TnPCB could not grow on biphenyl, and PCB degradation was inhibited in the presence of biphenyl. The most extensive surfactant and PCB degradation was achieved by the use of both recombinant strains together in the absence of biphenyl. PCB (Aroclor 1242) and surfactant (POL) concentrations were reduced from 25 ppm and 2000 ppm, respectively, to 6.5 ppm and 225 ppm, without the accumulation of surfactant degradation products. Given the inherent complexity of commercial surfactant preparations, the use of recombinant consortia to achieve extensive surfactant and PCB degradation appears to be an environmentally acceptable and effective PCB remediation option.

Molecular diagnostics and chemical analysis for assessing biodegradation of polychlorinated biphenyls in contaminated soils.

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), and 14CO2 evolution from 14C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to the bph gene probes, their ability to produce the yellow meta-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.

Cometabolic oxidation of polychlorinated biphenyls in soil with a surfactant-based field application vector.

Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.

Molecular diagnostics for polychlorinated biphenyl degradation in contaminated soils.

Molecular diagnostic methods using DNA hybridization with specific gene probes are being developed for the monitoring of microbial populations capable of polychlorinated biphenyl (PCB) degradation in contaminated soils. Evaluation of composite samples from contaminated electrical substation soil by gas chromatography (GC) indicated that the PCBs present in the soil (approximately 200 ppm) resulted from contamination with Aroclor 1248. The PCBs have been weathered or degraded so that the lower molecular weight PCB congeners are no longer present. Microbiological and molecular site characterizations are in progress to determine the abundance of PCB degradative organisms and catabolic genes present. Cloned DNA fragments for the bphC gene (2,3-dihydroxybiphenyl dioxygenase) from the biphenyl/chlorobiphenyl degradative pathways of different organisms were used as gene probes to identify indigenous microorganisms with bphC gene sequences. In colony hybridization experiments, positive signals with the pDA251 gene probe were detected in cultures from both contaminated and uncontaminated soils. The degradative abilities of indigenous microorganisms and an added PCB-degradative bacterial strain were also monitored with [14C]4-chlorobiphenyl mineralization assays and gas chromatography of PCB residues extracted from the soils. Enrichment of the contaminated soil with biphenyl and chlorobiphenyls did not stimulate the indigenous microorganisms to degrade the soil PCB. Nevertheless, enrichment of the contaminated soil with biphenyl and chlorobiphenyl and addition of the PCB-degrading strain Alcaligenes eutrophus GG4202 did result in additional degradation of the soil PCB. The results obtained from these experiments should assist in developing and monitoring a remediation plan for these PCB-contaminated soils.

Development of field application vectors for bioremediation of soils contaminated with polychlorinated biphenyls.

Field application vectors (FAVs), which are a combination of a selective substrate, a host, and a cloning vector, have been developed for the purpose of expressing foreign genes in nonsterile, competitive environments in which the gene products provide no advantage to the host. Such gene products are exemplified by the enzymes for the cometabolism of polychlorinated biphenyls (PCBs) through the biphenyl degradation pathway. Attempts to use highly competent PCB-cometabolizing strains in the environment in the absence of biphenyl have not been successful, while the addition of biphenyl is limited by its human toxicity and low water solubility. Broad-substrate-specificity PCB-degradative genes (bphABC) were cloned from a naturally occurring isolate. Pseudomonas sp. strain ENV307, into broad-host-range plasmid pRK293. The resulting PCB-degrading plasmids were transferred to the FAV host Pseudomonas paucimobilis 1IGP4, which utilizes the nontoxic, water-soluble, nonionic surfactant Igepal CO-720 as a selective growth substrate. Plasmid stability in the recombinant strains was determined in the absence of antibiotic selection. PCB-degrading activity was determined by resting cell assays. Treatment of contaminated soil (10, 100, or 1,000 ppm of Aroclor 1242) by surfactant amendment (1.0% [wt/wt]Igepal CO-720 in wet soil) and inoculation with recombinant isolates of strain 1IGP4 (approximately 4 x 10(6) cells per g of soil) resulted in degradation of many of the individual PCB congeners in the absence of biphenyl.(ABSTRACT TRUNCATED AT 250 WORDS)

Development and use of field application vectors to express nonadaptive foreign genes in competitive environments.

Many potential applications of genetically engineered microorganisms in environmental and agricultural biotechnology involve introducing genetic capabilities into nonsterile competitive environments in which they provide no advantage to the host. Field application vectors have been designed for the purpose of creating a temporary niche for the host in such environments. This technique involves the addition to the target environment of a selective substrate readily utilizable by the host microorganism but unavailable to most indigenous species. Thirteen nonionic and anionic detergents, representing a wide range of structural complexities and molecular weights, were screened as potential selective substrates. Competition experiments in soil, using Warburg respirometry, indicated that isolates from six different detergent enrichment cultures were more active on their corresponding detergents than the indigenous microorganisms. Detergents of intermediate structural complexities and molecular weights were most effective for use as selective substrates. A field application vector that utilizes 1.0% Igepal CO-720 (detergent) as the selective substrate and Pseudomonas paucimobilis 1IGP4 as the host was tested for its ability to increase the presence of nonadaptive tetracycline resistance marker genes in soil. In soil amended with the selective substrate, strain 1IGP4 plate counts increased by three orders of magnitude and tetracycline-resistant transformant (pRK293) counts increased from 1.8 x 10/g of soil to 4.3 x 10/g in 2 days. Inoculation in the absence of substrate amendment or amendment with a nonselective substrate did not result in growth of strain 1IGP4. These results demonstrate the effectiveness of field application vectors for increasing the concentration of nonadaptive genes in competitive environments.