Prediction of Substrate Removal Rates of Attached Microorganisms and of Relative Contributions of Attached and Suspended Communities at Field Sites

A mathematical model composed of a direct proportionality relationship between bulk water velocities and field-determined second-order microbial transformation rate coefficients, and the relative rate coefficient of a benchmark chemical, was developed for estimating the substrate removal rates of rapidly degraded chemicals by attached organisms in shallow (<1 m deep) aquatic ecosystems. Data from 31 field experiments involving the addition of 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) in nine field areas were used to determine a field-derived second-order rate coefficient for microbial transformation of the ester. By using 2,4-DME as a benchmark chemical, the model was used to predict microbial transformation rates of the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE) at five other field sites. The predicted half-lives of 2,4-DBE varied 1,500-fold and were within about a threefold range or less of the measured half-lives. Under conditions of mass transport limitation, the contributions of attached microorganisms relative to total microbial activities at various field sites were related to the ratio of water velocity, U, and depth, D, showing that historical definitions of ecosystems according to flow and depth characteristics are also valid for describing the process-related structure of ecosystems. An equation was developed for predicting the relative contributions of attached and suspended communities with values of U and D for lotic and lentic ecosystems. On the basis of this equation, attached microorganisms were expected to be insignificant in deep lentic ecosystems and suspended microorganisms were expected to be insignificant in shallow lotic systems for the same process carried out by both populations. Neglecting epiphytic microorganisms, both suspended and attached organisms were expected to be significant in wetlands.     

Predicting 2,4-Dichlorophenoxyacetic Acid Ester Transformation Rates in Periphyton-Dominated Ecosystems

Using batch cultures, we determined transformation rate coefficients for microbial transformation of 2,4-dichlorophenoxyacetic acid butoxyethyl ester (2,4-DBE) in periphyton-dominated ecosystems. Rates of 2,4-DBE loss were measured over short periods of time (usually less than 10 h), and first-order transformation rate coefficients (k₁) were determined under the specific conditions of low 2,4-DBE concentrations and no growth. Values for k₁ were divided by total plate counts and by biomass measured as ash-free dry weight to give second-order rate coefficients (k_b and k_AFDW, respectively) for use in predictive models. Using periphyton attached to Teflon strips, we also determined second-order rate coefficients based on the ratio of colonized surface area to container volume (k_A). Mean second-order rate coefficients were used to predict 2,4-DBE transformation rates in microcosms having diverse chemical and biological environments. The observed transformation rates among the microcosms were most accurately predicted by using k_A.     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

Effects of microbial community interactions on transformation rates of xenobiotic chemicals.

The effects of culture filtrates, mixed populations, and common microbial exudates on bacterial transformations of three agricultural and industrial chemicals were investigated. Test chemicals included methyl parathion, diethyl phthalate, and 2,4-dichlorophenoxyacetic acid butoxyethyl ester. The presence of various cultures, filtrates, or exudates of algae, fungi, or other bacteria either stimulated or inhibited bacterial transformation rates. Inhibition resulted from treatments that lowered the pH, and stimulation resulted from an increase in cell biomass (based on plate counts) and from a different process whereby rates of transformation per bacterial cell rapidly increased as much as 10-fold.     

Treating Soil Solution Samplers To Prevent Microbial Removal of Analytes

Soil microorganisms colonizing soil water sampling devices (lysimeters) reduced concentrations of biodegradable organic chemicals, including 2,4-dichlorophenoxyacetic acid methyl ester, alachlor, methyl m-chlorobenzoate, and metolachlor as water entered through porous ceramic cups. In some cases, losses exceeded 99%. Additions of either a biocide (sodium hypochlorite) or a bacteriostat (copper salt) prevented microbial activity so that concentrations of test chemicals inside lysimeters equaled those outside. Field studies further indicated that treating lysimeters with a copper salt effectively prevented microbial activity. Thus, chemically treating soil water samplers could improve the accuracy of soil water data for a wide variety of analytes, including environmentally important organics, such as pesticides and industrial wastes, and inorganics, such as ammonia and nitrate.     

Quantitative assessment of the effects of metals on microbial degradation of organic chemicals.

Biodegradation inhibition of a benchmark chemical, 2,4-dichloro-phenoxyacetic acid methyl ester (2,4-DME), was used to quantify the inhibitory effects of heavy metals on aerobic microbial degradation rates of organic chemicals. This procedure used lake sediments and aufwuchs (floating mats) collected in the field or from laboratory microcosms. Effects of CuCl2, HgCl2, ZnCl2, Cd(NO3)2, and Cr(NO3)3 at initial concentrations ranging from 0.3 microM to 73 mM (approximately 0.1 to 10,000 mg liter-1) were investigated. In general, such metallic compounds appeared to be considerably more inhibitory to the biodegradation of an organic chemical than high concentrations of microbially toxic organics studied previously. Effects of various metal concentrations were evaluated based on the following: (i) estimated MICs, (ii) concentrations that caused a significant effect on biodegradation parameters (both a greater than 10% decrease in Vmax and a greater than 10% increase in t1/2 for 2,4-DME degradation), and (iii) concentrations that caused biodegradation half-life doublings (HLDs). The MICs of metals in sediment were lowest for Zn2+ (0.10 microM) and highest for Cd2+ and Cu2+ (0.9 and 1.2 microM, respectively). The MICs of metals in aufwuchs were lowest for Hg2+ (0.01 microM), intermediate for Cu2+ and Zn2+ (0.42 and 0.62 microM, respectively), and highest for Cr3+ and Cd2+ (3.4 and 5.6 microM, respectively). Compared with Cu2+ on aufwuchs, 70 times more Zn2+, 250 times more Cr3+, and 1,000 times more Cd2+ was required to significantly affect aufwuchs biodegradation rate parameters and coefficients (Vmax and t1/2). Aufwuchs was significantly affected by the lowest Hg2+ concentration tested (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4).

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.     

Microbial diversity, tolerance, and biodegradation potential of urban wetlands with different input regimes

Though microbial transformations are the primary mechanism of contaminant attenuation in wetlands, much remains to be known about microbial communities in urban wetlands. In this study, the microbial communities from urban wetlands with different runoff regimes (i.e., a contaminated remnant wetland, a constructed wetland, and a remnant wetland) were assessed for their capacity to attenuate and tolerate typical urban runoff pollutants. Results from denaturing gradient gel electrophoresis of 16S rRNA genes showed relatively high similarity in community composition among the wetlands. Community-level physiological profiles had similar results but exhibited within-site variation in both the contaminated remnant and remnant wetlands. All wetland communities were less tolerant to copper than 2,4-dichlorophenoxyacetic acid; however, the contaminated remnant wetland had the highest tolerance. All study wetlands had a limited capacity to biodegrade model chlorinated aromatic compounds (e.g., 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate). Though having different input regimes and contaminant exposure histories, the study wetlands were generally similar with respect to microbial community diversity and function. Additionally, the generally low capacity for these wetlands to biodegrade mobile chlorinated organic contaminants offers preliminary insight into the limited ecosystem services these wetlands may provide in urban environments.     

Kinetic Concepts for Measuring Microbial Rate Constants: Effects of Nutrients on Rate Constants

We investigated the effect of preincubation of environmental waters amended with inorganic nutrients (nitrogen, phosphorus, and traces of iron and magnesium) on the kinetics of the microbial transformation of phenol, propanil, propyl ester of (2,4-dichlorophenoxy)acetic acid, methyl parathion, Ronnel, and methoxychlor in pond and river waters. No effect on the second-order rate constants for these compounds was observed, although there was an increase in the bacterial populations and the pseudo-first-order rate constants. The use of nutrient-amended waters could be a useful tool for estimating second-order rate constants for an expanded number of compounds. This technique would provide a larger data base for predicting the behavior of xenobiotic compounds in the environment by using currently available mathematical models.     

Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene

We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted.     

Quantitative distribution and metabolism of auxin herbicides in roots

The internal concentrations of four auxin herbicides— 2,4-dichlorophenoxyacetic acid, dicamba, picloram, and naphthaleneacetic acid—were measured in the roots of treated pea seedlings. Intact seedlings were immersed in solutions of labeled herbicides at concentrations sufficient to produce toxic symptoms (inhibition of elongation, radial enlargement, and lateral root proliferation). Measurements of volume and herbicide content of segments taken sequentially along the root showed that an acropetal concentration gradient of each herbicide was established within the root immediately following treatment. Although there was a net loss of herbicide in the following 24 hours, the gradient was maintained. Initially, the concentration of herbicide in the root tips exceeded that in the external medium.     

Humic substance-mediated reduction of iron(III) oxides and degradation of 2,4-D by an alkaliphilic bacterium,Corynebacterium humireducens MFC-5

With the use of an alkaliphilic bacterium, Corynebacterium humireducens MFC-5, this study investigated the reduction of goethite (α-FeOOH) and degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) mediated by different humic substances (humics) and quinones in alkaline conditions (pH of 9.0). The results indicated that (i) using sucrose as the electron donor, the strain MFC-5 was capable of reducing anthraquinone-2,6-disulfonic acid (AQDS), anthraquinone-2-disulfonic acid (AQS), anthraquinone-2-carboxylic acid (AQC), humic acid (HA) and fulvic acid (FA), and its reducing capability ranked as AQC > AQS > AQDS > FA > HA; (ii) the anaerobic reduction of α-FeOOH and 2,4-D by the strain was insignificant, while the reductions were greatly enhanced by the addition of quinones/humics serving as redox mediators; (iii) the Fe(III) reduction rate was positively related to the content of quinone functional groups and the electron-accepting capacities (EAC) of quinones/humics based on fourier-transform infrared spectroscopy (FT-IR) and electrochemical analyses; however, such a relationship was not found in 2,4-D degradation probably because quinone reduction was not the rate-limiting step of quinone-mediated reduction of 2,4-D. Using the example of α-FeOOH and 2,4-D, this study well demonstrated the important role of humics reduction on the Fe(III)/Fe(II) biogeochemical cycle and chlorinated organic compounds degradation in alkaline reducing environments.Funding Information This study was supported by the National Natural Science Foundation of China (Nos 41101211, 31070460, 41101477), and The Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.     

Biodegradation of methyl parathion in the presence of goethite: The effect of Pseudomonas sp. Z1 adhesion

In this research, the influence of goethite on biodegradation kinetic of methyl parathion was investigated in the presence of Pseudomonas sp. Z1. Semipermeable membrane experiments were performed to demonstrate the role of adhesion of degrading bacteria to surface of goethite in biodegradation of methyl parathion. Sorption of methyl parathion and bacteria onto goethite particles were also measured to assess the distribution of methyl parathion and bacteria between water and goethite surface. The first-order degradation rate constant of methyl parathion in different concentrations of goethite was in the order of 0.1 g L⁻¹ > 0.01 g L⁻¹ > 0 g L⁻¹ > 1 g L⁻¹ > 20 g L⁻¹, suggesting the presence of low concentrations of goethite accelerated the biodegradation of methyl parathion and high concentrations of goethite inhibited this biodegradation process. According to the result of semipermeable membrane experiment, when no bacterial attachment occurred in the system, the promotive effect of 0.1 g L⁻¹ goethite for microbial degradation was disappeared and the inhibition effect of 20 g L⁻¹ goethite increased. The results clearly demonstrated that the adhesion of bacteria to goethite was beneficial to the biodegradation of methyl parathion. The information obtained is of fundamental significance for the understanding of microbial degradation of organic pollution in soil.     

Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid

The agronomic performance of broad leaved crop plants such as cotton would be greatly improved if genetically-engineered resistance to broadleaf herbicides could both protect the plants from accidental spray drift damage and allow the suppression of problem broadleaf weeds by chemical means. Followingin vitro modification and the addition of plant expression signals, the gene for 2,4-D monooxygenase, a bacterial enzyme that degrades the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced into cotton plants byAgrobacterium-mediated transformation. First generation homozygous progeny of regenerated transgenic cotton plants carrying this gene exhibited up to a 50–100 fold increase in tolerance to 2,4-D compared with untransformed controls, and glasshouse trials suggest that the genetically-engineered plants would be completely protected from spray drift of 2,4-D, at least up to the normal field application rates commonly used on neighbouring cereal crops.     

Reductive transformation of methyl parathion by the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC7120

Organophosphorus compounds are toxic chemicals that are applied worldwide as household pesticides and for crop protection, and they are stockpiled for chemical warfare. As a result, they are routinely detected in air and water. Methods and routes of biodegradation of these compounds are being sought. We report that under aerobic, photosynthetic conditions, the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p-nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Methyl parathion transformation was not affected by mutations in the genes involved in nitrate reduction in cyanobacteria.     

Contact toxicity of some chemical and biological pesticides to several insect parasitoids and predators

Eight pesticides; methyl parathion, malathion (organo-phosphates), toxaphene (chlorinated hydrocarbon), carbaryl (carbamate), pyrethrin (plant derivative),Bacillus thuringiensis, nuclear polyhedrosis virus (Heliothis) (microbial insecticides), and 2,4-DB (postemergence herbicide) were evaluated at the minimum recommended field dose and reduced dosages for contact toxicity toBrachymeria intermedia (Hymenoptera: Chalcididae), Campoletis sonorensis (Hymenoptera: Ichneumonidae), Chelonus blackburni (Hymenoptera: Braconidae), Meteorus leviventris (Braconidae), Voria ruralis (Diptera: tachninidae), Chrysopa carnea (Neuroptera: Chrysopidae), andHippodamia convergens (Coleoptera: Coccinellidae). At minimum field dosages, percent mortality of parasitoids and predators was>27%, for the chemical insecticides. Mortality from pyrethrin was <31%, in all cases and 0% for 5 of the 8 species tested. Mortality of parasitoids and predators exposed toB. thuringiens is and NPV was<4% while mortality from 2,4-DB was<7%. The toxicity of chemical insecticides to parasitoids and predators at reduced dosages in increasing order of toxicity was malathion > carbaryl > toxaphene > methyl parathion.     

Reductive transformation of parathion and methyl parathion by Bacillus sp.

Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences and BIOLOG test, a soil bacterium was identified as Bacillus sp. DM-1. Using either growing cells or a cell-free extract, it transformed parathion and methyl parathion to amino derivatives by reducing the nitro group. Pesticide transformation by a cell-free extract was specifically inhibited by three nitroreductase inhibitors, indicating the presence of nitroreductase activity. The nitroreductase activity was NAD(P)H-dependent, O₂-insensitive, and exhibited the substrate specificity for parathion and methyl parathion. Reductive transformation significantly decreased the toxicity of pesticides.     

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells

Using suspension-cultured Acer pseudoplatanus cells requiring 2,4-dichlorophenoxyacetic acid for growth, the dependence of the population doubling time and the maximum increase in cell population density on the auxin concentration was studied. It appears that in the range of 2,4-dichlorophenoxyacetic acid concentration from 4 × 10⁻⁸ to 4 × 10⁻⁶ M, the rate of cell division during the logarithmic growth phase is independent of the auxin concentration, while the maximum number of cell generations obtained is limited by the initial auxin concentration. The significance of these two aspects of auxin action are discussed.     

Effect of Concentration of Organic Chemicals on Their Biodegradation by Natural Microbial Communities

The effect of concentration on the biodegradation of synthetic organic chemicals by natural microbial communities was investigated by adding individual ¹⁴C-labeled organic compounds to stream water at various initial concentrations and measuring the formation of ¹⁴CO₂. The rate of degradation of p-chlorobenzoate and chloroacetate at initial concentrations of 47 pg/ml to 47 μg/ml fell markedly with lower initial concentrations, although half or more of the compound was converted to CO₂ in 8 days or less. On the other hand, little mineralization of 2,4-dichlorophenoxyacetate and 1-naphthyl-N-methylcarbamate, or the naphthol formed from the latter, occurred when these compounds were present at initial concentrations of 2 to 3 ng/ml or less, although 60% or more of the chemical initially present at higher concentrations was converted to CO₂ in 6 days. It is concluded that laboratory tests of biodegradation involving chemical concentrations greater than those in nature may not correctly assess the rate of biodegradation in natural ecosystems and that low substrate concentration may be important in limiting biodegradation in natural waters.