Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848     

Earthworm egg capsules as vectors for the environmental introduction of biodegradative bacteria.

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2, 4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10(2) or 10(5) CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10(4) CFU R. eutropha (pJP4) g-1 of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2, 4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil previously enriched with organic substrates

Samples of chernozem soil were enriched with vanillic acid, protocatechuic acid glucose, a mixture of glucose and (NH₄)₂SO₄ (C∶N = 5∶1), ethanol and 2,4-dichlorophenoxyacetic acid (2,4-D). After a 6-d (with 2,4-D 35-d) incubation during which primary oxidation of the introduced substrates occurred, the soil was supplied with a solution of 2-¹⁴C-2,4-D (50ppm; 6.7kBq) and production of¹⁴CO₂ (product of microbial degradation of 2,4-D) was measured. Previously enriched samples exhibited a higher degradation rate; both the lag phase and doubling time of mineralization activity in the exponential phase of the process were markedly higher. This reflected an overall proliferation of bacteria and the increased relative proportion of bacterial strains capable of mineralizing 2,4-D in enriched samples. The stimulation of 2,4-D degradation may involve specific adaptation and selection mechanisms (as in the case with samples previously enriched with 2,4-D or its structural analogues—aromatic monomers, ethanol) as well as nonspecific mechanisms. The extent of mineralization of 2,4-D was not affected by soil pretreatment, about 1/3 of introduced radioactive carbon being invariably transformed to¹⁴CO₂.     

Dual-bioaugmentation strategy to enhance remediation of cocontaminated soil.

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 microg ml(-1), these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. 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%. Isolates Arthrobacter strain D9 and Pseudomonas strain 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-microg 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 microg of cadmium ml(-1) in pure culture and up to 60 microg 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 microg of cadmium g(-1). Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of¹⁴CO₂ a product of microbial degradation of 2-¹⁴C-2,4-D, was measured during 38-d incubation at 28°C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH₄)₂SO₄ (C:N=5∶1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization, did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent, of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into¹⁴CO₂). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers. The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.     

两株真降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植权的降解率分别为７４．４％和９５．０％;在固太发酵中植酸的降解率为４０％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、ｐＨ和水分等影响因了也进行了初步的探讨。经初步鉴定,这两株菌中有一株为拟青霉（Ｐａｅｃｉｌｏｍｙｃｅｓ ｓｐ）,另一株为青霉（Ｐｅｎｉｃｉｌｌｉｕ     

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2, 4–dichlorophenoxyacetate

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.     

Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10² or 10⁵ CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10⁴ CFU R. eutropha (pJP4) g⁻¹ of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

The influence of moisture on microbial transport, survival and 2,4-D biodegradation with a genetically marked Burkholderia cepacia in unsaturated soil columns

The influence of moisture on the survival, movement anddegradation activity of a 2,4-D degrading bacterium,Burkholderia cepacia strain BRI6001L, geneticallyengineered to contain bioluminescent and lactoseutilization genes, was studied in unsaturated soil columns.The distance traveled by BRI6001L was dependent on theclay content of the soil, higher clay contents beingresponsible for higher filtration coefficients. Long termsurvival, in excess of one year, was attributed to strainBRI6001L's ability to survive dry conditions. Changes inthe 2,4-D biodegradation rate showed a better correlationwith the BRI6001L population density than with the totalviable bacterial population. At moisture levels betweenfield capacity and 40% moisture (– 33 kPa to –100 kPa)2,4-D degradation was attributed mainly to BRI6001L. Atmoisture levels between 6 and 15%, 2,4-D disappearancewas attributed to the indigenous microbial population,with no degradation occurring at moisture levels below6%. Returning the moisture to above 40% led to anincrease of 4 orders of magnitude in the BRI6001Lpopulation density and to a 10-fold increase in the 2,4-Ddegradation rate. The ability to monitor a specificmicrobial population using reporter genes hasdemonstrated the importance of controlling moisturelevels for maximizing biodegradation rates in unsaturatedsoil environments.     

植被退化对温带典型草原根系-土壤系统碳氮分配的影响

以3种不同退化程度的温带典型草原(大针茅轻度退化、中度退化和重度退化)为研究对象,研究植被退化对温带典型草原土壤及根系碳氮含量及储量的影响。结果显示:(1)植被退化对地下根系碳含量影响不显著(P0.05),而对地下根系氮含量的影响显著(P0.05),中度退化样地根系氮含量显著高于轻度退化和重度退化样地(P0.05)。(2)植被退化对根系碳氮储量影响显著(P0.05),根系碳氮储量随着土层深度增加而减少,总根系碳氮储量随退化程度加剧而降低。(3)土壤有机碳、总碳和总氮含量及储量均受退化程度和采样深度的影响显著(P0.05),其含量随着土壤深度的增加而显著减少,随退化程度加剧而显著降低(P0.05)。(4)土壤是根系-土壤系统碳氮储存的最主要场所,储量占比90%以上。虽然土壤碳氮储量均存在表层聚集现象,但表层储量所占比例在各样地间差异显著(P0.05)。     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inoculant strain and organic matter content on kinetics of 2,4-dichlorophenoxyacetic acid degradation in soil.

We monitored rates of degradation of soluble and sorbed 2,4-dichlorophenoxyacetic acid (2,4-D) in low-organic-matter soil at field capacity amended with 1, 10, or 100 micrograms of 2,4-D per g of wet soil and inoculated with one of two bacterial strains (MI and 155) with similar maximum growth rates (mu max) but significantly different half-saturation growth constants (Ks). Concentrations of soluble 2,4-D were determined by analyzing samples of pore water pressed from soil, and concentrations of sorbed 2,4-D were determined by solvent extraction. Between 65 and 75% of the total 2,4-D was present in the soluble phase at equilibrium, resulting in soil solution concentrations of ca. 8, 60, and 600 micrograms of 2,4-D per ml, respectively. Soluble 2,4-D was metabolized preferentially; this was followed by degradation of both sorbed (after desorption) and soluble 2,4-D. Rates of degradation were comparable for the two strains at soil concentrations of 10 and 100 micrograms of 2,4-D per g; however, at 1 microgram/g of soil, 2,4-D was metabolized more rapidly by the strain with the lower Ks value (strain MI). We also monitored rates of biodegradation of soluble and sorbed 2,4-D in high-organic-matter soil at field capacity amended with 100 micrograms of 2,4-D per g of wet soil and inoculated with the low-Ks strain (strain MI). Ten percent of total 2,4-D was present in the soluble phase, resulting in a soil solution concentration of ca. 30 micrograms of 2,4-D per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new concept for reduction of diffuse contamination by simultaneous application of pesticide and pesticide-degrading microorganisms

Pesticide residues and their transformation products are frequently found in groundwater and surface waters. This study examined whether adding pesticide-degrading microorganisms simultaneously with the pesticide at application could significantly reduce diffuse contamination from pesticide use. Degradation of the phenoxyacetic acid herbicides MCPA (4-chloro-2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) was studied in soil microcosm experiments after simultaneous spraying of herbicide and herbicide-degrading bacteria on an agricultural soil and on a sand with low degradation potential. The latter represented pesticide use on non-agricultural soils poor in microbial activity. Degradation and possible loss of herbicidal effect were also tested in a system with plants and the amounts of bacteria needed to give satisfactory MCPA-degradation rate and the survival of degrading bacteria in formulated MCPA were determined. The results showed >80–99% degradation of 2,4-D and MCPA in soil within 1 day and >99% within 3 days after inoculation with 10⁵–10⁷ herbicide-degrading bacteria g⁻¹ dry weight of soil. Enhanced degradation of MCPA was also obtained in the presence of winter wheat and white mustard without loss of the intended herbicidal effect on white mustard. The survival of an isolated MCPA-degrading Sphingomonas sp. in three realistic concentrations of formulated MCPA was very poor, showing that in practical applications direct contact between the microorganisms and the pesticide formulation must be precluded. The applicability and economic feasibility of the method and the information needed to obtain a useable product for field use are discussed.     

Field-scale remediation of atrazine-contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase

We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms. This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA. AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. Soil contaminated by an accidental spill of atrazine (up to 29 000 p.p.m.) supported significant populations of indigenous microorganisms capable of atrazine catabolism. Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation. A subsequent field-scale study consisting of nine (0.75 m³) treatment plots was designed to test four treatment protocols in triplicate. Control plots contained moistened soil; biostimulation plots received 300 p.p.m. phosphate; bioaugmentation plots received 0.5% (w/w) killed, recombinant E. coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells. After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E. coli cells, and 77% in combination plots. In contrast, atrazine levels in control and biostimulation plots did not decline significantly. These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine.     

Response of the Soil Microbial Community to Changes in Precipitation in a Semiarid Ecosystem

Microbial communities regulate many belowground carbon cycling processes; thus, the impact of climate change on the structure and function of soil microbial communities could, in turn, impact the release or storage of carbon in soils. Here we used a large-scale precipitation manipulation (+18%, −50%, or ambient) in a piñon-juniper woodland (Pinus edulis-Juniperus monosperma) to investigate how changes in precipitation amounts altered soil microbial communities as well as what role seasonal variation in rainfall and plant composition played in the microbial community response. Seasonal variability in precipitation had a larger role in determining the composition of soil microbial communities in 2008 than the direct effect of the experimental precipitation treatments. Bacterial and fungal communities in the dry, relatively moisture-limited premonsoon season were compositionally distinct from communities in the monsoon season, when soil moisture levels and periodicity varied more widely across treatments. Fungal abundance in the drought plots during the dry premonsoon season was particularly low and was 4.7 times greater upon soil wet-up in the monsoon season, suggesting that soil fungi were water limited in the driest plots, which may result in a decrease in fungal degradation of carbon substrates. Additionally, we found that both bacterial and fungal communities beneath piñon pine and juniper were distinct, suggesting that microbial functions beneath these trees are different. We conclude that predicting the response of microbial communities to climate change is highly dependent on seasonal dynamics, background climatic variability, and the composition of the associated aboveground community.     

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.     

Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors

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. coli strain 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 the Burkholderia 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.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.