Quantification of Cryptosporidium parvum in natural soil matrices and soil solutions using qPCR.

Traditional microscopy methods for the detection and quantification of Cryptosporidium parvum in soil matrices are time-consuming, labor-intensive, and lack sensitivity and specificity. This research focused on developing a qPCR protocol for the sensitive and specific detection and quantification of C. parvum in natural soil matrices and soil-water extracts. The physico-chemical parameters - lysis media, number of thermal shocks and thawing temperatures - controlling DNA extraction efficiency were investigated. Experimental results identified oocyst age as a critical parameter affecting oocyst disruption and quantification. The most efficient oocyst disruption method for C. parvum oocysts regardless of their age was established as 5 thermal shocks with thawing at 65°C in Tris-EDTA (TE) buffer. In addition to the purification columns used to remove PCR inhibitors present in environmental matrices, a combination of 3mM MgCl(2) and 600ng/µl BSA yielded the highest amplicon yield for both young and aged oocysts. Sucrose flotation was determined to be a better oocyst isolation method than two-phase flotation. The optimized parameters for DNA extraction and the qPCR assay resulted in very specific and sensitive detection of C. parvum. Minimum detection limits were 0.667 for young C. parvum oocysts and 6.67 for aged C. parvum oocysts per PCR reaction. The accuracy of the detections and quantifications was 0.999. Protocol performance was tested in contrasting soil samples and soil-water extract samples on the basis of percentage of recovery (PR) values. Depending on the number of oocysts used to inoculate the samples, the average PR values ranged from 7.2 to 43.5%, 29.3-52.5%, and 11.5-60.8% for Trenton, Greenson, and Sparta soil-water extracts, respectively, and 12.1-77% for DI water. PR values ranged from 4.3% to 107.8% for Trenton, Greenson and Sparta soil samples.

Effectiveness of a Florida landfill biocover for reduction of CH4 and NMHC emissions.

Methane-oxidizing "biocovers" were constructed at the Leon County Landfill (Florida). The primary goal was to determine if a biocover placed above the existing thin (15 cm) intermediate clay cover would be capable of mitigating CH(4) and nonmethane hydrocarbon (NMHC) emissions to the atmosphere in this subtropical environment. A secondary goal was to maximize the use of locally recycled materials for biocover construction. The biocovers consisted of 30 or 60 cm of ground garden waste placed over a 15 cm gas distribution layer (clean crushed recycled glass from discarded fluorescent lights). The deep biocover reduced methane fluxes relative to the controls during temporal monitoring over more than a year; in large part, these reductions were attributable to increased methane oxidation. Both the shallow and the deep biocover exhibited significant percentages of negative fluxes (uptake of atmospheric methane) relative to the nonbiocover controls which had consistently positive fluxes. The overall annual effectiveness/performance of the biocover was limited by seasonally high moisture contents and the thin gas distribution layer. For NMHCs, the deep biocover demonstrated substantial reductions for nonmethane hydrocarbon emissions with high percentages of negative fluxes for several hydrocarbon groups, especially the aromatics, alkanes, and lower chlorinated compounds. Ranges of measured NMHC emissions (10(-9) to 10(-3) g m(-2) d(-1)) were similar to previous studies in the literature. Conservative calculations based on field data for total NMHC emissions from the 60 cm biocover area indicate that current U.S. Environmental Protection Agency (EPA) regulatory methods overestimate emissions by more than 2 orders of magnitude, suggesting that improved field-validated methods are needed.

Methane oxidation in landfill cover soils, is a 10% default value reasonable?

We reviewed literature results from 42 determinations of the fraction of methane oxidized and 30 determinations of methane oxidation rate in a variety of soil types and landfill covers. Both column measurements and in situ field measurements were included. The means for the fraction of methane oxidized on transit across the soil covers ranged from 22 to 55% from clayey to sandy material. Mean values for oxidation rate ranged from 3.7 to 6.4 mol m(-2) d(-1) (52-102 g m(-2) d(-1)) for the different soil types. The overall mean fraction oxidized across all studies was 36% with a standard error of 6%. The overall mean oxidation rate across all studies was 4.5 mol m(-2) d(-1) +/- 1.0 (72 +/- 16 g m(-2)d(-1)). For the subset of 15 studies conducted over an annual cycle the fraction of methane oxidized ranged from 11 to 89% with a mean value of 35 +/- 6%, nearly identical to the overall mean. Nine of these studies were conducted in north Florida at 30 degrees N latitude and had a fraction oxidized of 27 +/- 4%. Five studies were conducted in northern Europe ( approximately 50-55 degrees N) and exhibited an average of 54 +/- 14%. One study, conducted in New Hampshire, had a value of 10%. The results indicate that the fraction of methane oxidized in landfill greater than the default value of 10%. Of the 42 determinations of methane oxidation reported, only four report values of 10% or less.

Effect of temperature and oxidation rate on carbon-isotope fractionation during methane oxidation by landfill cover materials.

The quantification of methane oxidation is one of the major uncertainties in estimating CH4 emissions from landfills. Stable isotope methods provide a useful field approach for the quantification of methane oxidation in landfill cover soils. The approach relies upon the difference between the isotopic composition of oxidized gas at the location of interest and anaerobic zone CH4 and knowledge of alpha(ox), a term that describes the isotopic fractionation of the methanotrophic bacteria in their discrimination against (13)CH4. Natural variability in alpha(0x) in different landfill soils and the effect of temperature and other environmental factors on this parameter are not well defined. Therefore, standard determinations of alpha(ox), batch incubations of landfill cover soils with CH4, were conducted to determine alpha(ox) under a variety of conditions. When these results were combined with those of previous landfill incubation studies, the average alpha(ox) at 25 degrees C was 1.022 +/- 0.0015. alpha(ox) decreased with increasing temperature (-0.00039 alpha(ox) degrees C(-1)) overthe temperature range of 3-35 degrees C. alpha(ox) was found to be higher when determined after CH4-free storage and declined following CH4 pretreatment. alpha(ox) declined nonlinearly with increasing methane oxidation rate, Vmax.

Use of a biologically active cover to reduce landfill methane emissions and enhance methane oxidation.

Biologically-active landfill cover soils (biocovers) that serve to minimize CH4 emissions by optimizing CH4 oxidation were investigated at a landfill in Florida, USA. The biocover consisted of 50 cm pre-composted yard or garden waste placed over a 10-15 cm gas distribution layer (crushed glass) over a 40-100 cm interim cover. The biocover cells reduced CH4 emissions by a factor of 10 and doubled the percentage of CH4 oxidation relative to control cells. The thickness and moisture-holding capacity of the biocover resulted in increased retention times for transported CH4. This increased retention of CH4 in the biocover resulted in a higher fraction oxidized. Overall rates between the two covers were similar, about 2g CH4 m(-2)d(-1), but because CH4 entered the biocover from below at a slower rate relative to the soil cover, a higher percentage was oxidized. In part, methane oxidation controlled the net flux of CH4 to the atmosphere. The biocover cells became more effective than the control sites in oxidizing CH4 3 months after their initial placement: the mean percent oxidation for the biocover cells was 41% compared to 14% for the control cells (p<0.001). Following the initial 3 months, we also observed 29 (27%) negative CH4 fluxes and 27 (25%) zero fluxes in the biocover cells but only 6 (6%) negative fluxes and 22 (21%) zero fluxes for the control cells. Negative fluxes indicate uptake of atmospheric CH4. If the zero and negative fluxes are assumed to represent 100% oxidation, then the mean percent oxidation for the biocover and control cells, respectively, for the same period would increase to 64% and 30%.

Methane flux and oxidation at two types of intermediate landfill covers.

Methane emissions were measured on two areas at a Florida (USA) landfill using the static chamber technique. Because existing literature contains few measurements of methane emissions and oxidation in intermediate cover areas, this study focused on field measurement of emissions at 15-cm-thick non-vegetated intermediate cover overlying 1-year-old waste and a 45-cm-thick vegetated intermediate cover overlying 7-year-old waste. The 45 cm thick cover can also simulate non-engineered covers associated with older closed landfills. Oxidation of the emitted methane was evaluated using stable isotope techniques. The arithmetic means of the measured fluxes were 54 and 22 g CH(4) m(-2)d(-1) from the thin cover and the thick cover, respectively. The peak flux was 596 g m(-2)d(-1) for the thin cover and 330 g m(-2)d(-1) for the thick cover. The mean percent oxidation was significantly greater (25%) at the thick cover relative to the thin cover (14%). This difference only partly accounted for the difference in emissions from the two sites. Inverse distance weighing was used to describe the spatial variation of flux emissions from each cover type. The geospatial mean flux was 21.6 g m(-2)d(-1) for the thick intermediate cover and 50.0 g m(-2)d(-1) for the thin intermediate cover. High emission zones in the thick cover were fewer and more isolated, while high emission zones in the thin cover were continuous and covered a larger area. These differences in the emission patterns suggest that different CH(4) mitigation techniques should be applied to the two areas. For the thick intermediate cover, we suggest that effective mitigation of methane emissions could be achieved by placement of individualized compost cells over high emission zones. Emissions from the thin intermediate cover, on the other hand, can be mitigated by placing a compost layer over the entire area.

Methane oxidation in water-spreading and compost biofilters.

This study evaluated two biofilter designs to mitigate methane emissions from landfill vents. Water-spreading biofilters were designed to use the capillarity of coarse sand overlain by a finer sand to increase the active depth for methane oxidation. Compost biofilters consisted of 238-L barrels containing a 1:1 mixture (by volume) of compost to expanded polystyrene pellets. Two replicates of each type of biofilter were tested at an outdoor facility. Gas inflow consisted of an approximately 1:1 mixture (by volume) of CH4 and CO2. Methane output rates (J(out); g m(-2) day(-1)) were measured using the static chamber technique and the Pedersen et al. (2001) diffusion model. Methane oxidation rate (J(ox); g m(-2) day(-1)) and fraction of methane oxidized (f(ox)) were determined by mass balance. For methane inflow rates (J(in)) between 250 and 500 g m(-2) day(-1), the compost biofilter J(ox), 242 g m(-2) day(-1), was not significantly different (P = 0.0647) than the water-spreading biofilter J(ox), 203 g m(-2) day(-1); and the compost f(ox), 69%, was not significantly different (P = 0.7354) than water-spreading f(ox), 63%. The water-spreading biofilter was shown to generally perform as well as the compost biofilter, and it may be easier to implement at a landfill and require less maintenance.