Characterization of enterococci populations collected from a subsurface flow constructed wetland.

AIMS: The aim of this study was to identify and characterize the population of Enterococcus sp. in domestic wastewater as it flows through a constructed wetland. METHODS AND RESULTS: Four hundred and eighty-four Enterococcus isolates were collected from the inlet, various sites within and from the outlet of a plastic lined constructed wetland in College Station, TX. The wetland treated septic tank effluent that passed sequentially through two 1.89 m(3) septic tanks and a 1.89 m(3) pump tank allowing 48 l doses at a 24 l min(-1) rate. The Enterococcus isolates were identified to species using the commercial Biolog system. The 484 Enterococcus isolates were comprised of ten different species, including Enterococcus faecalis (30.6%), Enterococcus pseudoavium (24.0%), Enterococcus casseliflavus (12.8%), Enterococcus faecium (11.2%), Enterococcus mundtii (7.9%), Enterococcus gallinarum (6.2%), Enterococcus dispar (3.7%), Enterococcus hirae (2.1%), Enterococcus durans and Enterococcus flavescens both 0.8%. Of the 88 isolates collected from the inlet, only 9.1% of the isolates were identified as Ent. faecalis and Ent. pseudoavium (36.4%) was identified as the predominant species. Whereas of the 74 isolates collected from the outlet, the predominant species were identified as Ent. faecalis (29.7%). Species identification varied among sites within the wetland, but often Ent. faecalis was the predominant species. CONCLUSIONS: Our data suggest that while Ent. faecalis is the predominant species of Enterococcus found in domestic wastewater, the populations may shift during treatment as the wastewater flows through the constructed wetland. SIGNIFICANCE AND IMPACT OF THE STUDY: We found that shifts in Enterococcus species composition occurred during domestic wastewater treatment. This has implications for the identification of faecal pollution based on the presence of specific bacterial types associated with domestic wastewater.

Characterization of enterococci populations in livestock manure using BIOLOG.

The BIOLOG system was used to generate knowledge of enterococci populations found in fresh and dry manure of livestock (cattle (Bos taurus), horse (Equus caballus), and sheep (Ovis aires)). Six-hundred and forty Enterococcus isolates from the host sources were observed as a combined fresh manure unit and a combined dry manure unit, E. casseliflavus and E. mundtii were predominant in fresh manure (36% and 35%, respectively) as well as in dry manure (51% and 28%, respectively). The other species were found at a frequency of less than 10%. A chi-square test of the two most predominant Enterococcus sp. indicated that there were some significant differences among the frequency of E. casseliflavus and E. mundtii in cattle and sheep, but not horse. Despite these differences, these two species were overwhelmingly predominant among all three livestock sources.

Numbers of fecal streptococci and Escherichia coli in fresh and dry cattle, horse, and sheep manure.

Livestock are known contributors to stream pollution. Numbers of fecal streptococci and Escherichia coli in manure naturally deposited by livestock in the field are needed for activities related to bacterial source tracking and determining maximum daily bacterial loading of streams. We measured populations of fecal streptococci and E. coli in fresh and dry manure from cattle (Bos taurus L.), horses (Equus caballus L.), and sheep (Ovis aires L.) on farms in southern Idaho. Populations of indicator bacteria in dry manure were often as high as that in fresh manure from horse and sheep. There was a 2 log10 drop in the population of fecal coliform numbers in dry cattle manure from cattle in pastures but not from cattle in pens. Bacterial isolates used in source tracking should include isolates from both fresh and dry manure to better represent the bacterial source loading of streams.

Effects of drying on nitrification activity in zeoponic medium used for long-term space missions.

One component of a proposed life support system is the use of zeoponic substrates, which slowly release NH4+ into "soil" solution, for the production of plants. Nitrifying bacteria that convert NH4+ to NO3- are among the important microbial components of these systems. Survival of nitrifying bacteria in dry zeoponic substrates is needed, because the substrate would likely be stored in an air-dry state between croppings. Substrate was enriched for nitrifying bacteria and allowed to air-dry in a laminar flow hood. Stored substrate was analyzed for nitrifier survivability by measuring nitrifier activity at the beginning, 3 days, 1, 2, and 3 weeks. After rewetting, activity was approximately 9 micrograms N g-1 h-1 regardless of storage time. Nitrification rates did not decrease during storage. It seems unlikely that drying between plantings would result in practical reductions in nitrification, and reinoculation with nitrifying bacteria would not be necessary.

Nitrification in a zeoponic substrate.

Clinoptilolite is a zeolite mineral with high cation exchange capacity used in zeoponic substrates that have been proposed as a solid medium for growing plants or as a fertilizer material. The kinetics of nitrification has not been measured for NH4+ saturated zeoponic substrate. Experiments were conducted to evaluate the production of NO2- and NO3-, and nitrifier populations in zeoponic substrates. Small columns were filled with zeoponic substrate inoculated with a commercial inoculum or soil enrichment culture of nitrifying bacteria. In addition to column studies, a growth chamber study was conducted to evaluate the kinetics of nitrification in zeoponic substrates used to grow radishes (Raphanus sativus L.). The zeoponic substrate provided a readily available source of NH4+, and nitrifying bacteria were active in the substrate. Ammonium oxidation rates in column studies ranged from 5 to 10 micrograms N g-1 substrate h-1, and NO2- oxidation rates were 2 to 9.5 micrograms N g-1 substrate h-1. Rates determined from the growth chamber study were approximately 1.2 micrograms N g-1 substrate h-1. Quantities of NH4+ oxidized to NO2- and NO3- in inoculated zeoponic substrate were in excess of plant up-take. Acidification as a result of NH4+ oxidation resulted in a pH decline, and the zeoponic substrate showed limited buffering capacity.

Ultraviolet disinfection of effluent from subsurface flow constructed wetlands.

Subsurface flow constructed wetlands are becoming increasingly common for on-site treatment of domestic wastewater. Before spray application, wetland effluent must be disinfected. Traditionally, tablet chlorination has been used, but an alternative is needed to increase dependability. Consequently, we investigated the use of ultraviolet light disinfection of effluent from constructed wetlands. Two low pressure (254 nm) and two medium pressure ultraviolet bulbs (190 to 400 nm) were used for disinfection. Upon installation, all units disinfected effluent successfully. After 2 weeks in operation, three of the four units did not reduce fecal coliform populations below 200 in 100 ml of effluent due to decreased light intensity from films that developed on the bulbs. One unit consistently disinfected water to populations lower than 200 in 100 ml for 1 year without maintenance. Ultraviolet disinfection, as utilized, was not consistently suitable for disinfection of effluent from subsurface flow constructed wetlands because of coatings that developed on the bulbs and blocked the light.

Effects of umbrella palms and wastewater depth on wastewater treatment in a subsurface flow constructed wetland.

On-site subsurface flow constructed wetlands are designed to provide secondary quality effluent. Plants and wetland volume are considered in their design. There have been no studies, however, comparing wastewater treatment at different wastewater depths, and plant effects in wetlands are not completely understood. Investigations were conducted on these variables using four wetland cells 228 m wide by 4.75 m long containing 1 to 5 cm diameter river rock. Cyperus alternifolius (umbrella palms) were planted in one cell, and side-by-side comparisons were made between the planted and a control cell. Side-by-side comparisons were also made between cells with equal surface areas and different depths. At best umbrella palms improved effluent 5-day biochemical oxygen demand (BOD5) by 8%, suspended solids by 6%, and did not improve fecal coliform or P wastewater quality in July. When ambient air temperatures were < or = 12 degrees C during December, plants did not improve most treatment parameters. They did, however, significantly improve NH4+ treatment even when ambient air temperatures were as low as 8 degrees C. Increasing wastewater depth enhanced fecal coliform die-off but did not reduce effluent considered when maximum NH4+ reduction is a BOD5, suspended solids, NH4+, or P in effluent. Umbrella palms should be treatment goal and it is not necessary to reduce other parameters. Surface area was more important to wastewater treatment than depth.

Water flow patterns in subsurface flow constructed wetlands designed for on-site domestic wastewater treatment.

Bypass flow in subsurface flow constructed wetlands, which may be related to several wetland characteristics, reduces detention time and may result in inadequately treated wastewater. Subsurface flow constructed wetlands, 2.3 m wide by 4.8 m long and containing a gravel matrix, were used to investigate the impact of wetland depth, inlet pipe location, loading volume, and plants on water flow. Flow patterns were determined using blue dye or bromide as tracers. The blue dye adsorbed to the gravel and was not an effective tracer for following water movement. Water dispersed as it flowed through the wetland, and approximately two pore volumes of added water were required to displace approximately 99% of the bromide tracer added as a pulse. In 17 and 25 cm deep wetlands, water flow was uniform with depth, and inlet depth had little influence on water flow patterns. Water flow in a 40 cm deep wetland was not uniform with depth for either inlet pipe placement. The presence of plants caused preferential water flow around root masses, thereby, limiting their potential to interface with wastewater. Water mixing by dispersion from the surface to deeper depths may enhance aeration. Bromide was first detected in effluent after only 0.5 pore volumes of tap water had been added. This indicates that detention time for some wastewater would be less than expected, since plug flow is usually assumed in subsurface flow constructed wetlands. This occurrence should be considered for time-dependent treatments, such as fecal coliform and biological oxygen demand reduction.

Treatment of domestic wastewater by subsurface flow constructed wetlands filled with gravel and tire chip media.

Subsurface flow constructed wetlands (SFCWs) are becoming increasingly common in on-site treatment of wastewater. Gravel is the most popular form of wetland fill medium, but tire chips provide more porosity, are less dense, and less expensive. This study determines the treatment efficiency of SFCWs filled with gravel or tire chip media to treat domestic wastewater. The influent and effluent of six SFCWs filled with tire chip medium and six SFCWs filled with gravel were monitored for 5 to 16 consecutive months. Parameters measured included pH, biochemical oxygen demand (BOD5), total and volatile suspended solids, NH4, P, and fecal and total coliforms. The only clear difference between medium types in wetland performance was for P. Soluble P in the effluent averaged 1.6 +/- 1.0 mg l(-1) in the tire chip-filled wetlands and 4.8 +/- 3.2 mg l(-1) in the gravel-filled wetlands. Most likely, Fe from exposed wires in shredded steel-belted tires complexed with P to create an insoluble compound. Tire chips may be a better fill medium for SFCWs than gravel because of higher porosity, lower cost, and greater reduction of P in effluent.

Surfacing of domestic wastewater applied to soil through drip tubing and reduction in numbers of Escherichia coli.

Drip tubing is a technology that is increasing in use. The effectiveness of such systems in distributing the wastewater uniformly through the soil matrix, providing adequate removal of bacteria from wastewater, and keeping wastewater from reaching the soil surface has not been adequately evaluated. Experiments were conducted at two sites that had used drip tubing for approximately 3 years. This 3-year-old drip tubing and newly installed tubing were used in this investigation. A solution containing Brilliant Blue FCF dye and Escherichia coli, at an approximate concentration of 1 x 10(6) cells ml(-1), was applied to the sites through drip emitters. Reduction i n Escherichia coli populations reaching the soil surface was generally less than 10%. The route of travel for the solution reaching the soil surface was consistently along preferential flow paths and not uniformly through the soil matrix. Instances of water reaching the soil surface for drip tubing installed at 15 cm was nearly 50%. Increasing burial of the drip tubing from 15 to 30 cm nearly eliminated water surfacing. A 31 per emitter dose of water, immediately following drip line installation later increased instances of water reaching the soil surface for drip tubing buried at 30 cm. The volume of water applied per dose had little effect on the number of times water reached the soil surface. Inherent soil structural characteristics limited the drip tubing's ability to uniformly distribute water and adsorb bacteria. Drip tubing installation to 30 cm may be an important practice to reduce public health hazards from the likelihood of wastewater surfacing.

Uptake of 15N by macrophytes in subsurface-flow wetlands treating domestic wastewater.

Constructed subsurface-flow wetlands are becoming more common for on-site treatment of domestic wastewater to provide secondary quality effluent. Macrophytes are generally added to wetlands to increase treatment efficiency but their role in reducing N content is controversial. Our investigation utilized two subsurface-flow wetlands to determine the efficiency of different macrophytes in uptake of 15N labeled ammonium sulfate. Macrophytes in Wetland 1 recovered 35% of the added N in their shoots but only 5% of the added N was recovered in the shoots and roots in Wetland 2. A major difference for the two wetlands was N and hydraulic loadings. Wetland 1 received 7.5 Kg N ha(-1)d(-1) and Wetland 2 received 16.9 Kg N ha(-1)d(-1). Retention time for Wetland 1 based on pore volumes was 2.9 d and for Wetland 2 it was 1.2 d. The retardation factor for NH4+ was approximately 2.5 for both wetlands and the breakthrough curves indicated lack of plug flow. The importance of macrophytes in taking up NH4 appeared to dependent on N and hydraulic loadings.

Influence of salinity on bioremediation of oil in soil.

Spills from oil production and processing result in soils being contaminated with oil and salt. The effect of NaCl on degradation of oil in a sandy-clay loam and a clay loam soil was determined. Soils were treated with 50 g kg(-1) non-detergent motor oil (30 SAE). Salt treatments included NaCl amendments to adjust the soil solution electrical conductivities to 40, 120, and 200 dS m(-1). Soils were amended with nutrients and incubated at 25 degrees C. Oil degradation was estimated from the quantities of CO(2) evolved and from gravimetric determinations of remaining oil. Salt concentrations of 200 dS m(-1) in oil amended soils resulted in a decrease in oil mineralized by 44% for a clay loam and 20% for a sandy-clay loam soil. A salt concentration of 40 dS m(-1) reduced oil mineralization by about 10% in both soils. Oil mineralized in the oil amended clay-loam soil was 2-3 times greater than for comparable treatments of the sandy-clay loam soil. Amending the sandy-clay loam soil with 5% by weight of the clay-loam soil enhanced oil mineralization by 40%. Removal of salts from oil and salt contaminated soils before undertaking bioremediation may reduce the time required for bioremediation.

Utilization of carbon substrates, electrophoretic enzyme patterns, and symbiotic performance of plasmid-cured clover rhizobia.

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.

Variability in Effectiveness of Rhizobia during Culture and in Nodules.

The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.

Nitrogen-15 partitioning within a three generation tiller sequence of the bunchgrass Schizachyrium scoparium: response to selective defoliation.

Nitrogen partitioning among three generations of tillers within the bunchgrass Schizachyrium scoparium var. frequens was investigated in a controlled environment as a potential mechanism of herbivory tolerance. Nitrogen-15 was transported from the labelled primary tiller generation to both shoots and roots of nondefoliated secondary and tertiary tiller generations within 24 h. Partial defoliation increased shoot nitrogen concentration of secondary and tertiary generation tillers by 110 and 120%, respectively, 24 h following defoliation. Shoot nitrogen concentration was preferentially increased by partial defoliation of tertiary generation tillers throughout the 120 h experimental period, but diminished to concentrations comparable to nondefoliated tillers within shoots of the secondary generation at 72 h. In contrast to nitrogen concentration, the total amount of nitrogen imported by secondary and tertiary generation tillers decreased 62 and 73%, respectively, 24 h following partial defoliation and did not attain values comparable to respective nondefoliated tillers. Consequently, preferential nitrogen concentration occurred in response to partial tiller defoliation without an increase in total nitrogen import based on the reduction in the total nitrogen requirement per tiller generation associated with defoliation. Estimates of both the total amount of nitrogen import and nitrogen concentration are necessary to accurately interpret the dynamics of intertiller nitrogen allocation.

Inoculant maturity influences survival of rhizobia on seed.

Survival of Rhizobium trifolii on seeds of arrowleaf clover (Trifolium versiculosum Savi) and subclover (Trifolium subterraneum L.) was affected by the maturity of peat-, vermiculite-, and charcoal-based inoculants. Ten times more rhizobia survived on seed 4 days after inoculation when inoculants were stored (cured) before being utilized as compared with uncured inoculants. Increasing the curing time of inoculants beyond 4 weeks had little effect on increasing survival of seed-applied rhizobia.

Survival of cowpea rhizobia in soil as affected by soil temperature and moisture.

Successful inoculation of peanuts and cowpeas depends on the survival of rhizobia in soils which fluctuate between wide temperature and moisture extremes. Survival of two cowpea rhizobial strains (TAL309 and 3281) and two peanut rhizobial strains (T-1 and 201) was measured in two soils under three moisture conditions (air-dry, moist (-0.33 bar), and saturated soil) and at two temperatures (25 and 35 degrees C) when soil was not sterilized and at 40 degrees C when soil was sterilized. Populations of rhizobia were measured periodically for 45 days. The results in nonsterilized soil indicated that strain 201 survived relatively well under all environmental conditions. The 35 degrees C temperature in conjunction with the air-dry or saturated soil was the most detrimental to survival. At this temperature, the numbers of strains T-1, TAL309, and 3281 decreased about 2 logs in dry soil and 2.5 logs in saturated soil during 45 days of incubation. In sterilized soil, the populations of all strains in moist soil increased during the first 2 weeks, but decreased rapidly when incubated under dry conditions. The populations did not decline under saturated soil conditions. From these results it appears that rhizobial strains to be used for inoculant production should be screened under simulated field conditions for enhanced survival before their selection for commercial inoculant production.

Enumeration and identification of nitrogen-fixing bacteria from forage grass roots.

Root-soil cores were collected from forage grasses growing in a subtropical region of Texas and tested for acetylene reduction activity. The population density of nitrogen-fixing bacteria was measured, using various media and incubation conditions. Bacteria were confirmed as nitrogen fixing, using the acetylene reduction assay, and were classified according to standard biochemical and cultural methods. The majority of the nitrogen-fixing bacteria isolated from roots were Enterobacter cloacae or Klebsiella pneumoniae. Root-associated, nitrogen-fixing bacteria were isolated from 21 of 24 root-soil cores. The population densities of nitrogen-fixing bacteria ranged from approximately 10 to 3 x 10 per g of root. Population density on roots was significantly correlated with the rate of acetylene reduction but the relationship was not linear.