Automated dead-end ultrafiltration of large volume water samples to enable detection of low-level targets and reduce sample variability.

AIMS: A Portable Multi-use Automated Concentration System (PMACS) concentrates micro-organisms from large volumes of water through automated dead-end ultrafiltration and backflushing. The ability to detect microbial targets from ground, surface and cooling tower waters collected using standard methods was compared with samples from the PMACS in this study. METHODS AND RESULTS: PMACS (100 l) and standard grab samples (100-500 ml) were collected from sites in Florida and South Carolina, USA. Samples were analysed for the presence of faecal indicator bacteria (FIB; ground and surface water) or Legionella pneumophila (Lp; cooling tower water). FIB were enumerated by growth on selective media following membrane filtration or in IDEXX defined substrate media. Lp cells were detected by direct fluorescence immunoassay using FITC-labelled monoclonal antibodies targeting serogroups 1, 2, 4 and 6. FIB were found in PMACS samples from ground and surface waters when their concentrations were below detection limits in grab samples. The concentrations of Lp in cooling tower samples collected over 5 months were more consistent in PMACS samples than grab samples. CONCLUSIONS: These data demonstrate that PMACS concentration is advantageous for water monitoring. FIB were detected in PMACS samples when their concentrations were below the detection limits of the standard methods used. PMACS processing provided more representative samples of cooling tower waters reducing sample variability during long-term monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the utility of PMACS processing for enhanced monitoring of water for low-level microbial targets and for reducing sample variability in long-term monitoring programmes.

Interactions among phosphate amendments, microbes and uranium mobility in contaminated sediments.

The use of sequestering agents for the transformation of radionuclides in low concentrations in contaminated soils/sediments offers considerable potential for environmental cleanup. This study evaluated the influence of three types of phosphate (rock phosphate, biological phosphate, and calcium phytate) and two microbial amendments (Alcaligenes piechaudii and Pseudomonas putida) on U mobility. All tested phosphate amendments reduced aqueous U concentrations more than 90%, likely due to formation of insoluble phosphate precipitates. The addition of A. piechaudii and P. putida alone were found to reduce U concentrations 63% and 31%, respectively. Uranium removal in phosphate treatments was significantly reduced in the presence of the two microbes. Two sediments were evaluated in experiments on the effects of phosphate amendments on U mobility, one from a stream on the Department of Energy's Savannah River Site near Aiken, SC and the other from the Hanford Site, a Department of Energy facility in Washington state. Increased microbial activity in the treated sediment led to a reduction in phosphate effectiveness. The average U concentration in 1 M MgCl(2) extract from U contaminated sediment was 437 microg/kg, but in the same sediment without microbes (autoclaved), the extractable U concentration was only 103 microg/kg. The U concentration in the 1 M MgCl(2) extract was approximately 0 microg/kg in autoclaved amended sediment treated with autoclaved biological apatite. These results suggest that microbes may reduce phosphate amendment remedial effectiveness.

Phytoextraction of lead from firing range soil by Vetiver grass.

Phytoextraction techniques utilizing a sterile strain of Vetiver grass (Vetiveria zizanoides) along with soil amendments were evaluated for removing lead and other elements such as Zn, Cu, and Fe from the soil of a 50-year old active firing range at the Savannah River Site (SRS). Lead-contaminated soil (300-4500 ppm/kg) was collected, dried, placed in pots, fertilized, and used as a medium for growing transplanted Vetiver grass plants in a greenhouse. The uptake of metals by the plants was evaluated in response to various fertilization and pre-harvest treatment schemes. Baseline metal concentrations in the soil of all pots were measured prior to planting and when the plants were harvested. Plants grew better when fertilized with Osmocote fertilizer in comparison to plants fertilized with 10-10-10 (NPK) fertilizer. Application of a chelating agent, EDTA, one week prior to harvest significantly increased the amount of lead that was phytoextracted. Lead concentrations of up to 1390-1450 ppm/kg in tissue samples were detected. Maximum Pb levels were observed in root tissues. The addition of non-lethal doses of a slow-release herbicide in combination with EDTA did not appear to further enhance phytoextraction or the translocation of Pb into shoots. The study indicated that the use of Vetiver grass coupled with the use of chelating soil amendments has considerable potential for use as a remedial strategy for lead-contaminated soils such as those associated with firing ranges.

Respirometry for assessing the biodegradation of petroleum hydrocarbons.

The respiration method using the Micro-Oxymax respirometer was applied to evaluate the bioremediation potential of hydrocarbon-contaminated soils in two biopiles at the oil refinery in Czechowice-Dziedzice, Poland. In biopiles 1 and 2, two different technologies, i.e., enhanced (engineered) bioremediation and monitored natural attenuation (MNA) were used, respectively. In biopiles 1 and 2, the bioremediation process lasted 6 years and 8 months, respectively. The biodegradation of petroleum hydrocarbons was evaluated on the basis of CO2 production and O2 uptake. The CO2 production and O2 consumption rates during hydrocarbon biodegradation were calculated from the slopes of cumulative curve linear regressions. The results confirmed the hydrocarbon biodegradation process in both biopiles. However, in biopile 2 the process was more effective compared to biopile 1. In biopile 2, the O2 consumption and CO2 production means were 3.37 and 2.4 milliliters per kilogram of soil (dry weight) per minute, respectively. Whereas, in biopile 1, the O2 consumption and CO2 production means were 1.52 and 1.07 milliliters per kilogram of soil (dry weight) per minute, respectively. The mean biodegradation rate for biopile 2 was two times higher--67 mg hydrocarbons kg d.w.(-1)day(-1) compared with biopile 1, where the mean was 30 mg hydrocarbons kg d.w.(-1)day(-l). The results were correlated with petroleum hydrocarbon concentrations and microbial activity measured by dehydrogenase assay.

Identification of Thiothrix unzii in two distinct ecosystems.

AIMS: Molecular procedures were used to identify Thiothrix spp. in biofilms from sulphide-rich waters in two distinct ecosystems. METHODS AND RESULTS: Biofilm samples were obtained from two groundwater-fed systems in central and northern Florida, including an artesian spring and municipal water tank. The 16S rDNA in each sample was directly amplified by polymerase chain reaction. CONCLUSIONS: Clonal libraries of biofilm 16S rDNA from each site contained rDNA sequences that were 99-99.5% similar to Thiothrix unzii. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of T. unzii in a natural system. Biofilm formation by Thiothrix spp. can cause fouling in groundwater processing equipment, including municipal water-processing facilities, agricultural irrigation systems and spring water bottling plant filters. Biofouling can have severe economic and human health impacts as it will influence flow rates and related water treatments. Characterization of specific fouling bacteria and their molecular ecology is essential for their regulation.

Use of molecular techniques in bioremediation.

In a practical sense, biotechnology is concerned with the production of commercial products generated by biological processes. More formally, biotechnology may be defined as "the application of scientific and engineering principles to the processing of material by biological agents to provide goods and services" (Cantor, 2000). From a historical perspective, biotechnology dates back to the time when yeast was first used for beer or wine fermentation, and bacteria were used to make yogurt. In 1972, the birth of recombinant DNA technology moved biotechnology to new heights and led to the establishment of a new industry. Progress in biotechnology has been truly remarkable. Within four years of the discovery of recombinant DNA technology, genetically modified organisms (GMOs) were making human insulin, interferon, and human growth hormone. Now, recombinant DNA technology and its products--GMOs are widely used in environmental biotechnology (Glick and Pasternak, 1988; Cowan, 2000). Bioremediation is one of the most rapidly growing areas of environmental biotechnology. Use of bioremediation for environmental clean up is popular due to low costs and its public acceptability. Indeed, bioremediation stands to benefit greatly and advance even more rapidly with the adoption of molecular techniques developed originally for other areas of biotechnology. The 1990s was the decade of molecular microbial ecology (time of using molecular techniques in environmental biotechnology). Adoption of these molecular techniques made scientists realize that microbial populations in the natural environments are much more diverse than previously thought using traditional culture methods. Using molecular ecological methods, such as direct DNA isolation from environmental samples, denaturing gradient gel electrophoresis (DGGE), PCR methods, nucleic acid hybridization etc., we can now study microbial consortia relevant to pollutant degradation in the environment. These techniques promise to provide a better understanding and better control of environmental biotechnology processes, thus enabling more cost effective and efficient bioremediation of our toxic waste and contaminated environments.

Use of the MIDI-FAME technique to characterize groundwater communities.

Fatty acid methyl ester (FAME) profiles were identified directly from groundwater microbial communities concentrated on and extracted with polycarbonate filters. The sensitivity of this direct extraction method was determined using pure cultures of Acinetobacter junii, Pseudomonas putida and Stenotrophomonas maltophilia. A minimum concentration of 107 cells filter-1 was required to identify the predominant fatty acids from each culture. However, at least 3.7 x 109 cells filter-1 were required to obtain fatty acid profiles that matched the signature profiles for pure cultures in a commercial database. While several saturated fatty acids (i.e. 14 : 0, 16 : 0, 18 : 0) were extracted from the polycarbonate filters, they were readily subtracted from microbial fatty acid profiles and did not interfere with the characterization of pure cultures or environmental samples. For the environmental samples, 3 l of groundwater from the Savannah River Site, Aiken, SC, (USA) contained sufficient biomass for direct extraction. A comparative analysis of FAME groundwater profiles demonstrated a qualitative difference among communities sampled from spatially discrete locations, while a groundwater well that was sampled at two time points showed strong similarities over time. Concentration of microbial biomass on polycarbonate filters coupled with the MIDI-FAME extraction of both biomass and filter was a useful technique to characterize microbial communities from groundwater.

Production of a cell wall-specific monoclonal antibody to Fibrobacter succinogenes.

A monoclonal antibody (mAb) was raised against Fibrobacter succinogenes and produced after fusion as ascites in BALB/c mice. An ELISA was used to test for specificity and sensitivity of the mAb to detect F. succinogenes. The mAb BD1 was tested for sensitivity and cross-reactivity in detecting F. succinogenes with ELISA. The lower limits for F. succinogenes detection in pure and mixed culture-using mAb BD1 with ELISA was 10(5) cells ml-1. Twenty-six other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA but none was detected. Electron micrographs of F. succinogenes cells with immunogold labelling showed that the mAb BD1 reacted exclusively with cell wall epitopes but not intracellular material, as confirmed by ELISA.

Biofouling of groundwater systems by Thiothrix spp.

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.

Detection of Salmonella enteritidis in eggs and chicken with enzyme-linked immunosorbent assay.

An ELISA previously developed for the rapid detection of Salmonella enteritidis (SE) in environmental samples was modified and applied to food samples. A sandwich ELISA was designed that employs affinity-purified rabbit polyclonal antibodies for the capture stage and highly specific monoclonal antibodies for the detection stage. Thirty-nine species of bacteria other than SE, including 32 Salmonella species, were included in cross-reactivity testing with ELISA. Results showed no reactivity with any species tested besides SE. Salmonella enteritidis was added to homogenized food samples (chicken skin, meat, and eggs) to test ELISA sensitivity. The lower limit for ELISA detection of SE was 10(4) cells/mL for pure cultures and in 10% meat (wt/vol), 10(5) cells/mL in 10% skin (wt/vol), and 10(7) cells/mL in 10% eggs (wt/vol). Salmonella enteritidis detection with ELISA was confirmed with the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) method. Results were obtained within 24 h for ELISA method compared to 96 h for the BAM procedure. Results show that sensitivity of ELISA can vary with the type of food tested for detection of SE.

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.

An enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium aldrichii in anaerobiodigesters.

Monoclonal antibodies (Mabs) against Clostridium aldrichii were prepared by in vivo and in vitro immunization with whole cells and produced after fusion as ascites in BALB/c mice. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Mabs to detect Cl. aldrichii. The lower limit for Cl. aldrichii detection in pure and mixed culture with ELISA was 10(5) cells ml-1. Twenty other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA, but none was detected. The ELISA was used for detection of Cl. aldrichii over a 16-month period in five mesophilic continuously-stirred tank reactors (CSTR) with wood, glucose, sludge or sorghum as substrates. The population of Cl. aldrichii in the poplar wood anaerobic digester effluent was 10(6)-10(7) cells ml-1 over that time. These numbers were confirmed by anaerobic microbiological methods. Results from the ELISA technique were obtained in 36 h vs 3 weeks for culture methods. It is concluded that the ELISA is a useful, time-saving method for identification, detection and quantification of Cl. aldrichii in axenic, mixed culture, and in complex undefined cultures such as those found in anaerobic digesters.

Technical note: a noninvasive procedure for measuring goat heart rates.

Heart rates were obtained simultaneously from FM radio transmitters and heart rate monitors externally mounted on unanesthetized and unrestrained mixed-breed goats. Data from transmitters were highly correlated (r = .92, P < .0001) with data from monitors and the percentage difference in heart rates between the two devices was less than that observed between animals. Analyses also revealed that radio transmitters provided a reliable, repeatable, and valid method for the noninvasive measurement of goat heart rates.

Detection of Salmonella enteritidis in environmental samples by monoclonal antibody-based ELISA.

We have developed a enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ASCII) for the detection of Salmonella enteritidis in environmental samples. ELISA was used to test for sensitivity and specificity of ASCII. 38 other species of bacteria, including 31 Salmonella species were included in cross-reactivity testing with ELISA. ASCII showed no reactivity with any other species tested. ASCII was found to be an IgG1 specific for S. enteritidis lipopolysaccharide (LPS). The lower limits for S. enteritidis detection was 10(5) cells/ml for pure cultures and in 10% sludge (w/v). Environmental samples (raw wastewater, wastewater effluents, mixed liquor and aerobically digested sludge) were obtained twice from five sites and ELISA tested for the presence of S. enteritidis. ELISA results compared to the American Public Health Association (APHA) method of Salmonella detection were not significantly different (P greater than 0.05). The ELISA took 24 h for completion compared to 96-120 h for the APHA procedure. Results demonstrate the reliability of the ELISA and, more importantly, provides a rapid means of detection of S. enteritidis in environmental samples.

Seasonal temperature and its influence on plasma corticosterone, triiodothyronine, thyroxine, plasma protein and packed cell volume in mature male chickens.

1. The relationship between seasonal changes in environmental temperature and hematological parameters was investigated in mature, single comb white leghorn (SCWL) male chickens. 2. Samples of blood plasma, obtained monthly from two groups of birds over two separate 12 month periods, were analysed for corticosterone (CT), 3,5,3'-triiodothyronine (T3), thyroxine (T4), plasma protein (PP), and packed cell volume (PCV). 3. Statistical analyses revealed that blood plasma concentrations of T3 were significantly correlated negatively with monthly dry-bulb temperatures. 4. There were no consistent or significant relationships between monthly dry-bulb temperature and CT, T4, PP or PCV over the two 12 month periods. 5. The results of this study indicate that blood plasma concentrations of T3 are influenced by season of year in mature, male domestic fowl.

Adrenal and body temperature changes in rabbits exposed to varying effective temperatures.

Eight adult New Zealand White rabbits were exposed individually, in series, to each of 23 effective temperatures (t(eff)) until body temperature (tb) increased 1.1 degrees C or for a period of 2 hours. Body temperature was measured to the nearest 0.1 degree C using FM radio transmitters in the pre-test (baseline) condition and at 2 minute intervals during the test conditions where t(eff) ranged between 21.7 and 34.7 degrees C. The frequency at which the rabbits displayed a 1.1 degree C rise in tb was related to the magnitude of the t(eff), with 100% of the rabbits manifesting this change at t(eff) greater than 30.2 degrees C. At t(eff) of 28.4 through 30.2 degrees C, some, but not all, of the rabbits showed a 1.1 degree C rise in tb whereas none displayed the 1.1 degree C rise in tb at t(eff) below 28.4 degrees C. The mean time necessary for the 1.1 degree C rise in tb was negatively correlated (P less than 0.01) to the magnitude of the t(eff). The significantly (P less than 0.01) elevated plasma corticosterone in rabbits exhibiting 0.6 degree C and 1.1 degree C rise in tb suggests that those animals were stressed physiologically by the experimental procedure. It is concluded that the conditions associated with increased tb induce physiological changes commonly associated with stressors and that the techniques reported herein should be useful in establishing upper environmental temperature limits for housing rabbits.

A simplified telemetry system for monitoring body temperature in small animals.

An inexpensive but reliable telemetry system for long-term, sequential monitoring of body temperature in up to 20 laboratory animals is described. The system consists of frequency-modulated (FM) temperature transmitters, remote-controlled power switches to extend battery life, a multi-channel telemetry receiver, and a frequency counter interfaced with a personal computer to record data. Analysis of body temperature data obtained from four New Zealand White rabbits confirms the reliability and value of this system.

A simple remote-controlled power switch for internalized bioelectronic instrumentation.

This communication presents a remote-controlled power switch for extending the battery life of biomedical instruments implanted into animals or humans. The switching action is controlled externally to the implant by an inductive link between two coils, one contained in the implant and one external to the implant. The external coil sends an electromagnetic pulse to the implant, triggering a CMOS "D" flip-flop connected as a toggle switch--its state is toggled on or off upon receiving the external pulse. The standby current drain of the switch is about 4 nA. The remote triggering range is approximately 20-50 cm. Testing of the switch, surgically implanted as part of a telemetry transmitter, is also discussed.

Feeding time and body temperature interactions in broiler breeders.

Four groups of 70-wk-old broiler breeder females were fed once daily at 0600, 1000, 1400, and 1800 h to determine the effect of feeding time and eating on body temperature. The photoperiod was from 0430 to 1930 h. Four floor pens of 30 hens each were assigned per feeding time. Following a 9-day adjustment period, body temperature was determined, in series, by rectal probe of 5 birds/pen at 7 and 3 h prefeeding and 1, 5, 9, and 13 h postfeeding. Body temperature was increased .5 C at 1 h postfeeding in all groups and at 5 h postfeeding in the 0600-h fed group. The rate of feed consumption was fastest with afternoon feeding. Four 1-yr-old broiler breeder males were implanted with an FM radio transmitter for monitoring body temperature and housed in an environmental control chamber. Body temperature was monitored when the birds were fed at 0600, 1000, 1400, and 1800 h. The chamber temperature cycled from 22.2 to 33.3 C (22.2 C: 2200 to 0800 h; 33.3 C: 1200 to 1600 h; 27.8 C: 0800 to 1200 h and 1600 to 2200 h). Lights were on from 0430 to 1930 h. Body temperature changes were also monitored under constant temperature (27.8 C) and light for birds fed ad libitum or at 1000 h. Body temperature increased as much as 1.5 C following feeding and reached a maximum at 5, 4, 3, and 2 h postfeeding at feeding times of 0600, 1000, 1400, and 1800 h, respectively. Males unable to feed displayed a significantly increased body temperature when they observed other birds eating. A specific body temperature response to feeding activity was observed only when males were fed once daily under constant environment.