Karen L. Josephson

Dual-Bioaugmentation Strategy To Enhance Remediation of Cocontaminated Soil

ABSTRACT Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 μg ml−1, these isolates represented the common soil genera Arthrobacter, Bacillus, andPseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. IsolatesArthrobacter strain D9 and Pseudomonasstrain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-μg ml−1 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 μg of cadmium ml−1 in pure culture and up to 60 μg of cadmium g−1 in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 μg of cadmium g−1. Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.

Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner

This two year study evaluated the prevalence of indicator bacteria and specific pathogens in10 ‘normal’ kitchens in the United States. In Phase I, none of the kitchens wascleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens weremonitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms andfaecal coliforms. Almost all locations at all households exhibited contamination, with the sink andsponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations insink and sponge samples were very high, with 63 and 67% of all samples being positive,respectively. Escherichia coli was detected in 16·7% of all sink surfaces and33·3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobialdisinfectant cleaner which families were encouraged to use but not forced to do so; in some cases,the product was used infrequently or not at all. This regimen did not demonstrate any consistentreduction in the incidence of bacterial contamination. By contrast, in the final phase of the studywhere disinfectant use was targeted for surfaces soon after contamination with foods or hands,the incidence of contamination decreased dramatically. These data show that normal kitchens caneasily be contaminated with a variety of bacterial contaminants including faecal coliforms, E.coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobialagents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likelyto reduce the incidence of bacterial contaminants.

A national study on the residential impact of biological aerosols from the land application of biosolids

Aims:  The purpose of this study was to evaluate the community risk of infection from bioaerosols to residents living near biosolids land application sites.

Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil.

ABSTRACT This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

ABSTRACT Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism,Escherichia coli ATCC 15224, by plate mating withRalstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 107 and 108 transconjugants g of dry soil−1 for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil−1, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genusBurkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.

Survival of indicator organisms in Sonoran Desert soil amended with sewage sludge

Abstract Anaerobically digested sewage sludge is currently applied to farmland surrounding Tucson, Arizona to supply nutrients for the growth of cotton. Bacterial pathogens introduced into the environment by this practice may survive or move through the soil profile thus posing health risks to animals and humans. We have conducted both laboratory and field studies to monitor the survival and transport of bacterial pathogens added to soil via sludge. In laboratory studies, sewage sludge was added to soil and incubated at constant moisture and temperature conditions. Populations of fecal streptococci, fecal coliforms and total coliforms were monitored at weekly intervals using the Most Probable Number (MPN) method. Two soils and 3 temperatures were evaluated. Field studies determined the survival of indicator organisms in the surface horizon, and survival and transport of fecal coliforms up to a depth of 300 cm. We found that soil moisture, texture and temperature all affected the persistence of bacterial p...

Estimated Occupational Risk from Bioaerosols Generated during Land Application of Class B Biosolids

Some speculate that bioaerosols from land application of biosolids pose occupational risks, but few studies have assessed aerosolization of microorganisms from biosolids or estimated occupational risks of infection. This study investigated levels of microorganisms in air immediately downwind of land application operations and estimated occupational risks from aerosolized microorganisms. In all, more than 300 air samples were collected downwind of biosolids application sites at various locations within the United States. Coliform bacteria, coliphages, and heterotrophic plate count (HPC) bacteria were enumerated from air and biosolids at each site. Concentrations of coliforms relative to Salmonella and concentrations of coliphage relative to enteroviruses in biosolids were used, in conjunction with levels of coliforms and coliphages measured in air during this study, to estimate exposure to Salmonella and enteroviruses in air. The HPC bacteria were ubiquitous in air near land application sites whether or not biosolids were being applied, and concentrations were positively correlated to windspeed. Coliform bacteria were detected only when biosolids were being applied to land or loaded into land applicators. Coliphages were detected in few air samples, and only when biosolids were being loaded into land applicators. In general, environmental parameters had little impact on concentrations of microorganisms in air immediately downwind of land application. The method of land application was most correlated to aerosolization. From this large body of data, the occupational risk of infection from bioaerosols was estimated to be 0.78 to 2.1%/yr. Extraordinary exposure scenarios carried an estimated annual risk of infection of up to 34%, with viruses posing the greatest threat. Risks from aerosolized microorganisms at biosolids land application sites appear to be lower than those at wastewater treatment plants, based on previously reported literature.

Specific detection of rhizobia in root nodules and soil using the polymerase chain reaction

Abstract The polymerase chain reaction (PCR) amplification of specific DNA sequences, allows specific and sensitive detection of bacteria at the genus, species or strain level depending on the design of the oligonucleotide primers. In this study we utilized 20 mer primers that flanked a 300 bp region of the npt II gene of the transposon Tn5 thus allowing for the amplification of this region. Insertion of the Tn 5 element into rhizobia allowed for detection of these cells using PCR amplification. Using the npt II -specific primers and Tn5 insertion mutants of Rhizobium leguminosarum bv. phaseoli we were able to detect these specific rhizobia strains in root nodules of bean plants and in inoculated soils. Utilization of genus-specific gene sequences would allow for estimates of cells of that genus in environmental samples. Conversely, use of gene sequences common to rhizobia, e.g. nif and nod sequences, would give estimates of the population of rhizobia. This paper serves to illustrate the use of PCR, for detecting gene sequences in an environmental sample such as a root nodule.

Functional establishment of introduced chlorobenzoate degraders following bioaugmentation with newly activated soil. Enhanced contaminant remediation via activated soil bioaugmentation.

Introduced degraders often do not survive when applied to polluted sites; however, the potential for successful bioaugmentation may be increased if newly activated soil (containing indigenous degrader populations recently exposed to the contaminant) or potentially active soil (containing indigenous degrader populations not previously exposed to the contaminant) is used as the inoculant. To investigate this concept, Madera and Oversite soils were amended with 0 or 500 micrograms of 2-, 3-, or 4-chlorobenzoate per gram soil. The Madera degraded 2-chlorobenzoate while the Oversite degraded 3- and 4-chlorobenzoate. After 22 days of incubation, non-active soils that had not degraded chlorobenzoate were bioaugmented with the appropriate activated soil that had been exposed to and degraded chlorobenzoate. Thus, Oversite soil that had not degraded 2-chlorobenzoate was bioaugmented with Madera soil that had degraded 2-chlorobenzoate. Likewise, Madera soil that had not degraded 3- or 4-chlorobenzoate was bioaugmented with the Oversite soil that had degraded 3- or 4-chlorobenzoate. Additionally, the non-active soils were bioaugmented with the corresponding potentially active soils. The Oversite soil amended with activated Madera soil degraded the 2-chlorobenzoate within 3 days of bioaugmentation. The Madera soil amended with activated Oversite soils degraded the 3- and 4-chlorobenzoate within 20 and 6 days, respectively. Large degrader populations developed in microcosms bioaugmented with activated soil, and shifts in the 3- and 4-CB degrader community structures occurred following bioaugmentation. In contrast, bioaugmentation with potentially active soil did not impact degradation. The results indicate the potential for bioaugmentation with newly activated soil to enhance contaminant degradation.

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture: Bioaugmentation effects on soil microorganisms

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 μg 3-chlorobenzoate g-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraderswere enumerated on minimal salts media containing 3-chlorobenzoate. Culturableheterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 μg 3-chlorobenzoate g-1 dry soil inoculated microcosms, degradersincreased from the initial inoculum and decreased following degradation of 3-CB.Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 107 colony-forming-units g-1 dry soil in the 500 μg 3-chlorobenzoate g-1 dry soil microcosms, but none were detected in the 1000 μg 3-chlorobenzoate g-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species.In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 μg 3-chlorobenzoate g-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.