M. B. Cassidy

Environmental applications of immobilized microbial cells: A review

Immobilized microbial cells have been used extensively in various industrial and scientific endeavours. However, immobilized cells have not been used widely for environmental applications. This review examines many of the scientific and technical aspects involved in using immobilized microbial cells in environmental applications, with a particular focus on cells encapsulated in biopolymer gels. Some advantages and limitations of using immobilized cells in bioreactor studies are also discussed.

Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

Chlorophenol and nitrophenol metabolism by Sphingomonas sp UG30.

Sphingomonas sp UG30 is a pentachlorophenol (PCP)-degrading bacterial strain capable of degrading several nitrophenolic compounds, including p-nitrophenol (PNP), 2,4-dinitrophenol (2,4-DNP), p-nitrocatechol and 4,6-dinitro-o-cresol (DNOC). The ability to degrade both chlorophenolic and nitrophenolic compounds is probably not restricted to UG30, but may also be possessed by other pentachlorophenol-degrading Sphingomonas spp. The interesting question arises as to whether there is any point of convergence between the initial pathways of PCP and nitrophenol degradation in these microorganisms. There is some experimental evidence that PCP-4-monooxygenase is involved in metabolism of both p-nitrophenol and 2,4-dinitrophenol. Further studies are needed to confirm this and to examine the role(s) of other PCP-degrading enzymes in nitrophenol metabolism by this microorganism. In this paper, we review some of the taxonomic, biochemical, physiological and ecological properties of Sphingomonas sp UG30 with respect to biodegradation of PCP and nitrophenolic compounds.

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants. The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists. The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.

A comparison of enumeration methods for culturable Pseudomonas fluorescens cells marked with green fluorescent protein

The detection of bacteria in environmental samples using genetic markers is valuable in microbial ecology. The green fluorescent protein (GFP) reporter gene was studied under nutrient starvation conditions at 4 degrees C, 23 degrees C and 30 degrees C in Pseudomonas fluorescens R2fG1 cells tagged with a red-shifted gfp. Fluorescence intensity was not significantly different in cells maintained in a buffer for at least 48 days at all the tested temperatures. gfp-Tagged R2fG1 cells were introduced into bulk soil microcosms and soil microcosms with wheat seedlings. GFP-marked cells were enumerated immediately after inoculation into soil and again in soil and root samples after 10 days. Counts of culturable colonies were obtained from drop plates using 5-microl aliquots of serial dilutions viewed with an epifluorescent microscope. Traditional spread plates (using 100-microl aliquots) and the most-probable-number (MPN) method using a spectrofluorometer were also used to enumerate the GFP-marked Pseudomonas cells in soil, rhizosphere and rhizoplane samples. Microcolonies were visualized on root surfaces under the epifluorescent microscope after immobilizing in agar and incubation for 24 h. Counts from traditional spread plates were significantly higher (P<0.05) than the population estimates of the MPN method for all treatments at any sampling time. Counts using the drop plate method, however, were not significantly different (P<0.05) except in one treatment, and provided similar estimates in half the time of spread plates and at an estimated third of the cost.

A Comparison of Five Bioassays to Monitor Toxicity During Bioremediation of Pentachlorophenol-Contaminated Soil

Five bioassays were used to measure toxicity during bioremediation of a soil contaminated with pentachlorophenol (PCP; 335 ppm), polycyclic aromatic hydrocarbons (PAHs; 1225 ppm) and petroleum hydrocarbons (19 125 ppm). Different bioremediation treatments were tested in soil microcosms including amendment with phosphorus and/or PCP-degrading Pseudomonas sp. UG30, either as free cells or encapsulated in κ-carrageenan. Soil toxicity was monitored using the solid-phase Microtox test, SOS-chromotest, lettuce seed germination, earthworm survival and sheep red blood cell (RBC) haemolysis assays. PCP levels were reduced in all treatments after 210 days. The RBC lysis assay, Microtox test and SOS-chromotest indicated reduced toxicity in most of the microcosms by day 210. Trends depicted by lettuce seed germination and earthworm survival LC50 values varied with each treatment. For example, in soil amended with phosphorus, both the seed germination and earthworm survival LC50 data suggested increased soil toxicity. However, for soil treated with encapsulated Pseudomonas sp. UG30 cells, the earthworm survival LC50 data indicated reduced toxicity while seed germination LC50 values showed little change from values obtained prior to bioremediation. Our results show that toxicity trends in a contaminated soil during bioremediation differ according to the assay used.

Pentachlorophenol biodegradation by Pseudomonas spp. UG25 and UG30

Eighty-nine bacterial isolates obtained by enrichment from pentachlorophenol (PCP)-contaminated soil samples were tested for PCP dechlorination activity and hybridization to pcpB (encoding PCP-4-monooxygenase) and pcpC (encoding tetrachlorohydroquinone reductive dehalogenase) gene probes synthesized by polymerase chain reaction from Flavobacterium sp. ATCC 39723 genomic DNA. Seven isolates were able to dechlorinate PCP, hybridize to both pcpB and pcpC DNA probes, and mineralize sodium pentachlorophenate (NaPCP) at an initial concentration of 100μg/ml. Although the seven PCP-mineralizing isolates possessed DNA sequences homologous to the Flavobacterium pcpB and pcpC genes, restriction analysis revealed sequence differences between the isolates and the Flavobacterium PCP dechlorination genes. Two isolates, designated UG25 and UG30, with the fastest onset and highest extent of PCP mineralization were selected for further study. Both isolates were tentatively identified as Pseudomonas spp. and exhibited stoichiometric release of Cl− ions as PCP was degraded. The release of Cl− began concomitantly with PCP disappearance from the medium. Both UG25 and UG30 degraded NaPCP at a concentration of 250 μg/ml in a minimal salt medium. Supplementation of the medium with glutamate increased the NaPCP degradation threshold of UG25 to a concentration of 300 μg/ml but did not affect that of UG30. 31P-NMR spectra of UG25 and UG30 cell suspensions exposed to PCP showed lower intracellular ATP levels and a more acidic cytoplasmic pH relative to untreated cells. This de-energization may explain the lack of cell growth in the presence of high PCP concentrations.

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

Abstract A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by 14CO2 evolution from radiolabelled [U-14C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur.

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A contaminated soil from Ontario, Canada containing 350–370 ppm pentachlorophenol (PCP) and 21000 ppm total petroleum hydrocarbons (TPH) was inoculated with PCP-degrading Pseudomonas sp UG30 cells either encapsulated in κ κ-carrageenan or as free cells. Uninoculated control soil produced 18.8 ± 3.9% of 14CO2 from 100000 dpm [U-14C]-PCP after 30 weeks incubation at 22°C. Addition of phosphate increased PCP mineralization, whereas addition of nitrogen inhibited mineralization almost completely in soil not inoculated with UG30 cells. No enhancement of mineralization was observed in soil with the addition of 108 CFU g−1 dry soil free UG30 cells. κ κ-Carrageenan-encapsulated UG30 cells at the same inoculum density mineralized 64.7 ± 0.3% PCP in soil after 26 weeks. Repeated inoculations with encapsulated UG30 cells up to six times over 6 weeks resulted in 64.8 ± 1.9% mineralization of radiolabelled PCP within 9 weeks, although by 12 weeks all treatments with encapsulated cells had mineralized to this level. Addition of sterile beads (controls) led to less than 16.6 ± 9.2% mineralization within 16 weeks. Varying initial inoculum densities of encapsulated cells were compared to determine effects on PCP mineralization. Cells grown inside the beads, and higher initial cell densities exhibited greater mineralization activity in the first weeks, but by 20 weeks PCP mineralization was approximately 70.0 ± 8.0% and was not significantly different between all soil treatments. Our results show encapsulation can enhance pollutant mineralization in a chemically contaminated soil.

Survival of lac-lux marked Pseudomonas aeruginosa UG2Lr cells encapsulated in κ-carrageenan and alginate

A milder method for encapsulating viable bacterial cells in κ-carrageenan was developed. A 1% (w/v) κ-carrageenan concentration amended with clay and skim milk was used as a lower-cost alternative to alginate for potential introductions of bacteria into soil. Survival of lac-lux marked Pseudomonas aeruginosa UG2Lr cells during long-term storage in dried κ-carrageenan and alginate beads at 4°C was compared. Survival of UG2Lr cells in the κ-carrageenan formulated beads at Log 8.3 CFU/g dry beads was significantly better in the first 90 days than that in alginate at Log 7.8 CFU/g dry beads. After 360 days survival remained stable at Log 7.8 CFU/g dry beads and was not significantly different between the two carriers. Electron micrographs revealed bacterial cells in the κ-carrageenan matrix, and degradation of the bead surface after 4 weeks incubation in non-sterile soil.