Ian L. Pepper

New approaches for bioaugmentation as a remediation technology

Bioaugmentation is commonly employed as a remediation technology. However, numerous studies indicate that introduced microorganisms often do not survive in the environment and thus do not increase contaminant remediation. This review details several new approaches that may increase the persistence and activity of exogenous microorganisms and/or genes following introduction into the environment. These techniques include: (1) bioaugmentation with cells encapsulated in a carrier such as alginate; (2) gene bioaugmentation where the goal is for the added inoculant to transfer remediation genes to indigenous microorganisms; (3) rhizosphere bioaugmentation where the microbial inoculant is added to the site along with a plant that serves as a niche for the inoculants growth; and (4) phytoaugmentation where the remediation genes are engineered directly into a plant for use in remediation without a microbial inoculant. Additionally, the review discusses the generation of genetically engineered microorganisms for use in bioaugmentation along with methods for the control of the engineered microorganisms in the environment, and the potential effects of the release on indigenous organisms. Various methods for the detection of introduced microorganisms such as real-time polymerase chain reaction (PCR) and reporter genes are also addressed. Ultimately, these new approaches may broaden the application of bioaugmentation as a remediation technology.

Detection of protozoan parasites and microsporidia in irrigation waters used for crop production

The occurrence of human pathogenic parasites in irrigation waters used for food crops traditionally eaten raw was investigated. The polymerase chain reaction was used to detect human pathogenic microsporidia in irrigation waters from the United States and several Central American countries. In addition, the occurrence of both Cryptosporidium oocysts and Giardia cysts was determined by immunofluorescent techniques. Twenty-eight percent of the irrigation water samples tested positive for microsporidia, 60% tested positive for Giardia cysts, and 36% tested positive for Cryptosporidium oocysts. The average concentrations in samples from Central America containing Giardia cysts and Cryptosporidium oocysts were 559 cysts and 227 oocysts per 100 liters. In samples from the United States, averages of 25 Giardia cysts per 100 liters and <19 (average detection limit) Cryptosporidium oocysts per 100 liters were detected. Two of the samples that were positive for microsporidia were sequenced, and subsequent database homology comparisons allowed the presumptive identification of two human pathogenic species, Encephalitozoon intestinalis (94% homology) and Pleistophora spp. (89% homology). The presence of human pathogenic parasites in irrigation waters used in the production of crops traditionally consumed raw suggests that there may be a risk of infection to consumers who come in contact with or eat these products.

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.

Microbial Responses to Environmentally Toxic Cadmium

A bstractWe analyzed the soil microbial communities from one uncontaminated and two metal-impacted soils and found that while cadmium adversely affected the numbers of culturable bacteria in all soils, cadmium-resistant isolates were found from each of the soils. With exposure to 24 and 48 μg ml-1 soluble cadmium, the metal-contaminated soil communities were more resistant than the uncontaminated soil community. In addition, in one metal-stressed soil, the resistant population became more resistant with increased cadmium levels. Ribosomal 16S DNA sequencing identified the isolates as Arthrobacter,Bacillus, or Pseudomonas spp. Further characterization demonstrated that two of the isolates were highly resistant to soluble cadmium with maximum resistance at 275 μg ml-1 cadmium. These isolates were also resistant to a variety of antibiotics, namely ampicillin, gentamicin, penicillin, and streptomycin, but no overall correlation was found between enhanced antibiotic resistance and cadmium resistance. One Pseudomonas isolate H1 did become more resistant with increasing cadmium levels, suggesting a different resistance mechanism at high cadmium concentrations.

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.

Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples.

The advent of the PCR has greatly enhanced our ability to detect human enteric viral pathogens in the environment, including water, municipal wastes, sewage, food, air, and fomites ([2][1], [3][2], [59][3], [69][4], [79][5]). This is especially true for those viruses which do not grow in cell

Hazards from Pathogenic Microorganisms in Land-Disposed Sewage Sludge

Sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are precipitated from wastewater and sewage during primary, secondary, and tertiary sewage treatment. Present in these sludges are significant numbers of microorganisms that include viral, bacterial, protozoan, fungal, and helminth pathogens. The treatment of sludge to reduce biochemical oxygen demand, solids content, and odor is not always effective in reducing numbers of pathogens. This becomes a public health concern because the infectious dose for some of these pathogens may be as low as 1 particle (virus) to 50 organisms (Giardia). When sludge is applied to land for agricultural use and landfill compost, these pathogens can survive from days (bacteria) to months (viruses) to years (helminth eggs), depending on environmental conditions. Shallow aquifers can become contaminated with pathogens from sludge and, depending on groundwater flow, these organisms may travel significant distances from the disposal site. Communities that rely on groundwater for domestic use can become exposed to these pathogens, leading to a potential disease outbreak. Currently, methods to determine the risk of disease from pathogens in land-disposed sludge are inadequate because the sensitivity of pathogen detection is poor. The application of recombinant DNA technology (gene probes and polymerase chain reaction) to environmental samples may provide increased sensitivity for detecting specific pathogens in land-disposed sludge and greatly improved risk assessment models for our exposure to these sources of pathogens.

Soil microbial activity as an indicator of soil fertility: Long-term effects of municipal sewage sludge on an arid soil

The utility of a selection of soil microbial assays for predicting the effects of land application of municipal sewage sludge on long-term soil fertility was evaluated in a 4 yr study with one unfertilized control plot, and plots with anaerobically digested sewage sludge applied at the optimal rate (based on N requirements) for cotton growth and three times the optimal rate. These loading rates were 8.0 and 24tha−1 yr−1 (dry wt) during 4 yr. The soil was a Pima clay loam (Typic Torrifluvent) which was planted to Upland cotton (Gossypium hirsutum L.). After 4 yr of annual sludge application and annual growth of cotton crops, it was found that sewage sludge application had had no significant effect on various measured soil physical and chemical properties other than an increase in available PO4-P. Total heavy metal contents were not affected by sludge application, but some DTPA-TEA-extractable metals (Zn, Cu, Pb and Ni) increased significantly with sludge treatment. Results also indicated that 4 yr of sewage sludge application had had no significant adverse effect on soil microbial populations or activity. Soil microbial activity was measured by viable heterotrophic plate counts for bacteria, actinomycetes and fungi; acridine orange direct counts for bacteria; the dehydrogenase assay and CO2 evolution. In some cases, there was a significant elevation of a few of the measured variables (dehydrogenase activity and CO2 evolution). Cotton lint yields in year 4 of the study were not significantly affected by treatment, but plant stand was significantly decreased with higher sludge application. As there was no significant association (Pearson product-moment correlation coefficient or the Kendall ρ b, as applicable) between measurements of microbial activity and cotton plant growth, this study lends no support to using soil microbial activity as measured here as a predictive index of soil fertility as affected by land application of sewage sludge. However, this study does illustrate that long-term applications of sludge to arid southwestern desert soils does not adversely affect microbial populations or activity.

Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors

ABSTRACT A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection,Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. colistrain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of theBurkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

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.