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In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

Animal manure is applied to agricultural land in areas of high livestock production. In the present study, we evaluated ageing of atrazine in two topsoils with and without addition of manure and in one subsoil. Ageing was assessed as the bioavailability of atrazine to the atrazine mineralizing bacteria Pseudomonas sp. strain ADP. Throughout an ageing period of 90 days bioavailability was investigated at days 1, 10, 32, 60 and 90, where ~10(8) cells g(-1) of the ADP strain was inoculated to the (14)C-atrazine exposed soil and (14)CO2 was collected over 7 days as a measure of mineralized atrazine. Even though the bioavailable residue decreased in all of the three soils as time proceeded, we found that ageing occurred faster in the topsoils rich in organic carbon than in subsoil. For one topsoil rich in organic carbon content, Simmelkær, we observed a higher degree of ageing when treated with manure. Contrarily, sorption experiments showed less sorption to Simmelkær treated with manure than the untreated soil indicating that sorption processes are not the only mechanisms of ageing. The other topsoil low in organic carbon content, Ringe, showed no significant difference in ageing between the manure-treated and untreated soil. The present study illustrates that not simply the organic carbon content influences adsorption and ageing of atrazine in soil but the origin and composition of organic matter plays an important role.

A molecular study on how the abundance of the dechlorinating culture KB-1 affects dechlorination rates in clay till is presented. DNA extracts showed changes in abundance of specific dechlorinators as well as their functional genes. Independently of the KB-1 added, the microbial dechlorinator abundance increased to the same level in all treatments. In the non-bioaugmented microcosms the reductive dehalogenase gene bvcA increased in abundance, but when KB-1 was added the related vcrA gene increased while bvcA genes did not increase. Modeling showed higher vinyl-chloride dechlorination rates and shorter time for complete dechlorination to ethene with higher initial concentration of KB-1 culture, while cis-dichloroethene dechlorination rates were not affected by KB-1 concentrations. This study provides high resolution abundance profiles of Dehalococcoides spp. (DHC) and functional genes, highlights the ecological behavior of KB-1 in clay till, and reinforces the importance of using multiple functional genes as biomarkers for reductive dechlorination., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations of tfdA in the range 1 × 10(5) to 5 × 10(7) gene copies g(-1) of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model.

Separate quantification of three classes of tfdA genes was performed using TaqMan quantitative real-time PCR for 13 different soils subsequent to mineralization of three phenoxy acids. Class III tfdA genes were found to be involved in mineralization more often than class I and II tfdA genes.

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g(-1) soil to approximately 3 x 10(4) genes g(-1) soil and to 7 x 10(5) genes g(-1) soil for topsoil initially added to 2.3 mg MCPA kg(-1) (dry weight) soil and 20 mg MCPA kg(-1) (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.

Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event, tfdA genes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations oí tfdA in the range 1 x 105 to 5 x 107 gene copies g-1 of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a higher tfdA gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model. [ABSTRACT FROM AUTHOR]

Abstract: Mineralization and sorption of 14C-ring labeled herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) were quantified along with the tfdA gene abundance in 7 different soils. The soils tested were five gravel soils from urban locations, one soil from the embankment of a railway track, and finally an agricultural soil as a control. The mineralization experiments were performed with a concentration of MCPA of 5 mg/kg and incubated at 10 °C for a period of 60 days. With K d values ranging from 0.04 to 0.41 l kg−1 the sorption experiments revealed that binding of MCPA to the six gravel soils was lower compared to the control soil which had a K d value of 0.91 l kg−1. The potential for MCPA mineralization varied from less than 5 to over 55% mineralized in 60 days. The most rapid MCPA mineralization was observed in the soil from the Danish railway tracks with 55% mineralized after only 18 days. The mineralization data was fitted to degradation kinetic models, which indicated that growth occurred as a response to MCPA degradation in most of the soils. Soil DNA was extracted and tfdA genes responsible for the first step in MCPA degradation were quantified by real-time PCR (qPCR) at appropriate time points throughout the mineralization experiments. Indicating growth of specific MCPA degraders, the abundance of class III tfdA genes showed an increase during MCPA mineralization in those soils able to mineralize MCPA. These findings emphasize the importance of the presence of microorganisms that are able to readily degrade MCPA, to avoid groundwater leaching following use on urban gravel areas that possess low binding ability of the compound. [Copyright &y& Elsevier]

Abstract: The allelochemical sorgoleone is produced in and released from the root hairs of sorghum (Sorghum bicolor). Studies have confirmed that it is the release of sorgoleone that causes the phytotoxic properties of sorghum, and sorgoleone has a potential to become a new natural herbicide, or the weed suppressive activity of sorghum can be utilized in integrated weed management. Since sorgoleone is released into soil, knowledge of the fate of sorgoleone in soil is essential if it is to be utilized as an herbicide. Fate studies will characterize the persistence and mobility of the compound. Three types of radioactively labelled sorgoleone were produced and used to study mineralization (complete degradation to CO2) of this lipid benzoquinone in four soils, two from the United States of America (Mississippi) and two from Denmark. The studies showed that sorgoleone was mineralized in all soils tested. The methoxy group of sorgoleone was readily mineralized, whereas mineralization of the remaining molecule was slower. Mineralization kinetics indicated that microorganisms in American soils were able to use sorgoleone as a source of energy. [Copyright &y& Elsevier]

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an α-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 105 cells g−1 soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg−1). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg−1, the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg−1 amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg−1 scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil. [ABSTRACT FROM AUTHOR]

Abstract: Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca2+ in combination with high pH. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach reveals a robust co-extraction protocol for a range of molecular analysis of diverse soil environments. [Copyright &y& Elsevier]