Nitrogen impacts on atrazine-degrading Arthrobacter strain and bacterial community structure in soil microcosms

The objective of this study was to investigate the impacts of exogenous nitrogen on a microbial community inoculated with the atrazine-degrading Arthrobacter sp. in soil amended with a high concentration of atrazine. Inoculated and uninoculated microcosms for biodegradation tests were constructed. Atrazine degradation capacity of the strain DAT1 and the strain's atrazine-metabolic potential and survival were assessed. The relative abundance of the strain DAT1 and the bacterial community structure in soils were characterized using quantitative PCR in combination with terminal restriction fragment length polymorphism. Atrazine degradation by the strain DAT1 and the strain's atrazine-metabolic potential and survival were not affected by addition of a medium level of nitrate, but these processes were inhibited by addition of a high level of nitrate. Microbial community structure changed in both inoculated and uninoculated microcosms, dependent on the level of added nitrate. Bioaugmentation with the strain DAT1 could be a very efficient biotechnology for bioremediation of soils with high concentrations of atrazine.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79–82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53–83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N2-ethyl-N?-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79-82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53-83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Simazine degradation in bioaugmented soil: urea impact and response of ammonia-oxidizing bacteria and other soil bacterial communities

The objective of this study was to investigate the impact of exogenous urea nitrogen on ammonia-oxidizing bacteria (AOB) and other soil bacterial communities in soil bioaugmented for simazine remediation. The previously isolated simazine-degrading Arthrobacter sp. strain SD1 was used to degrade the herbicide. The effect of urea on the simazine degradation capacity of the soil bioaugmented with Arthrobacter strain SD1 was assessed using quantitative PCR targeting the s-triazine-degrading trzN and atzC genes. Structures of bacterial and AOB communities were characterized using terminal restriction fragment length polymorphism. Urea fertilizer could affect simazine biodegradation and decreased the proportion of its trzN and atzC genes in soil augmented with Arthrobacter strain SD1. Bioaugmentation process could significantly alter the structures of both bacterial and AOB communities, which were strongly affected by urea amendment, depending on the dosage. This study could provide some new insights towards s-triazine bioremediation and microbial ecology in a bioaugmented system. However, further studies are necessary in order to elucidate the impact of different types and levels of nitrogen sources on s-triazine-degraders and bacterial and AOB communities in bioaugmented soil.     

Long-term dynamics of the atrazine mineralization potential in surface and subsurface soil in an agricultural field as a response to atrazine applications

The dynamics of the atrazine mineralization potential in agricultural soil was studied in two soil layers (topsoil and at 35-45 cm depth) in a 3 years field trial to examine the long term response of atrazine mineralizing soil populations to atrazine application and intermittent periods without atrazine and the effect of manure treatment on those processes. In topsoil samples, ¹⁴C-atrazine mineralization lag times decreased after atrazine application and increased with increasing time after atrazine application, suggesting that atrazine application resulted into the proliferation of atrazine mineralizing microbial populations which decayed when atrazine application stopped. Decay rates appeared however much slower than growth rates. Atrazine application also resulted into the increase of the atrazine mineralization potential in deeper layers which was explained by the growth on leached atrazine as measured in soil leachates recovered from that depth. However, no decay was observed during intermittent periods without atrazine application in the deeper soil layer. atzA and trzN gene quantification confirmed partly the growth and decay of the atrazine degrading populations in the soil and suggested that especially trzN bearing populations are the dominant atrazine degrading populations in both topsoil and deeper soil. Manure treatment only improved the atrazine mineralization rate in deeper soil layers. Our results point to the importance of the atrazine application history on a field and suggests that the long term survival of atrazine degrading populations after atrazine application enables them to rapidly proliferate once atrazine is again applied.     

Competitive biodegradation of dichlobenil and atrazine coexisting in soil amended with a char and citrate

The role of char nutrients in the biodegradation of coexisting dichlobenil and atrazine in a soil by their respective bacterial degraders, DDN and ADP, was evaluated. Under growing conditions, their degradation in soil extract was slow with <40% and <20% degraded within 64 h, respectively. The degradation in extracts and slurries of char-amended solids increased with increasing char content, due to nutritional stimulation on microbial activities. By supplementing soil extract with various major nutrients, the measured degradation demonstrated that P was the exclusive limiting nutrient. The reduction in the degradation of coexisting dichlobenil and atrazine resulted apparently from the competitive utilization of P by DDN and ADP. With a shorter lag phase, ADP commenced growing earlier than DDN with the advantage of utilizing P first in insufficient supply. This resulted in an inhibition on the growth of DDN and thus suppression on dichlobenil degradation.     

Chemotaxis to atrazine and detection of a xenobiotic catabolic plasmid in Arthrobacter sp. DNS10

Introduction

A plasmid named pDNS10 was detected from an atrazine-degrading strain Arthrobacter sp. DNS10 which has been isolated previously in our laboratory.

Materials and methods

In this paper, a special plasmid-detecting method and drop assays experiments were mainly used to achieve research goals.

Results and discussion

pDNS10 exhibited an excellent stability because it also could be detected even when the strain DNS10 has been subcultured under nonselective conditions for eight times. Over a 48-h incubation period, the OD₆₀₀ of samples inoculated with strain DNS10 and strain DNS10-ST (both of them contained pDNS10) were 0.31 ± 0.042 and 0.305 ± 0.034, respectively ,whereas the OD₆₀₀ of samples inoculated strain without pDNS10 (strain DNS10-PE) was only 0.138 ± 0.018. No atrazine was detected in the inoculated strain DNS10 and strain DNS10-ST samples at this period. Contrarily, the atrazine-degrading rate of strain DNS10-PE was only 5.23 ± 0.71%. Furthermore, both the two types of strains containing pDNS10 confirmed the presence of known degrading genes such as trzN, atzB, and atzC. It suggests that pDNS10 is an atrazine catabolic plasmid. In drop assays experiments, the wild-type strain DNS10 cells were chemotactically attracted to atrazine, whereas strain DNS10-PE showed no chemotaxis to atrazine and hydroxyatrazine. There was some relationship between atrazine degradation and the chemotactic response towards atrazine in strain DNS10.

Conclusions

The biochemical characteristics of pDNS10 and the chemotaxis characteristics of strain DNS10 could help us in better understanding of the mechanism of atrazine degradation by strain DNS10.     

The rhizosphere and PAH amendment mediate impacts on functional and structural bacterial diversity in sandy peat soil

To reveal the degradation capacity of bacteria in PAH polluted soil and rhizosphere we combined bacterial extradiol ring-cleavage dioxygenase and 16S rRNA analysis in Betula pubescens rhizoremediation. Characterisation of the functional bacterial community by RFLP revealed novel environmental dioxygenases, and their putative hosts were studied by 16S rRNA amplification. Plant rhizosphere and PAH amendment effects were detected by the RFLP/T-RFLP analysis. Functional species richness increased in the birch rhizosphere and PAH amendment impacted the compositional diversity of the dioxygenases and the structural 16S rRNA community. A shift from an Acidobacteria and Verrucomicrobia dominated to an Alpha- and Betaproteobacteria dominated community structure was detected in polluted soil. Clone sequence analysis indicated catabolic significance of Burkholderia in PAH polluted soil. These results advance our understanding of rhizoremediation and unveil the extent of uncharacterized functional bacteria to benefit bioremediation by facilitating the development of the molecular tool box to monitor bacterial populations in biodegradation.     

Impact of the novel neonicotinoid insecticide Paichongding on bacterial communities in yellow loam and Huangshi soils

Insecticides are widely sprayed in modern agriculture for ensuring the crop yield, which could also lead to contamination and insecticide residue in soils. Paichongding (IPP) is a novel neonicotinoid insecticide and was developed recently in China. Soil bacterial community, diversity, and community composition vary widely depending on environmental factors. As for now, little is known about bacterial species thriving, bacterial community diversity, and structure in IPP-spraying soils. In present study, IPP degradation in yellow loam and Huangshi soils was investigated, and bacterial communities and diversity were examined in soil without IPP spray and with IPP spray through pyrosequencing of 16S ribosomal RNA (rRNA) gene amplicons. The degradation ratio of IPP at 60 days after treatment (DAT) reached 51.22 and 34.01 % in yellow loam and Huangshi soil, respectively. A higher richness of operational taxonomic units (OTUs) was found in yellow loam soil (867 OTUs) and Huangshi soil (762 OTUs) without IPP spray while OUTs were relatively low in IPP-spraying soils. The community composition also differed both in phyla and genus level between these two environmental conditions. Proteobacteria, Firmicutes, Planctomycetes, Chloroflexi, Armatimonadetes, and Chlorobi were stimulated to increase after IPP application, while IPP inhibited the phyla of Bacteroidetes, Actinobacteria, and Acidobacteria.     

高效阿特拉津降解菌株DNSIO降解条件优化

从长期施用阿特拉津的寒地黑土耕层（0～10cm）土壤中筛选到一株能以除草剂阿特拉津为氮源生长的降解菌株，结合16SrRNA序列分析结果，将该菌株命名为Arthrobacter sp．DNSl0。在接种量为10。CFU／mL的条件下，菌株DNSl0在24h内对100mg／L阿特拉津的降解率为99．41％。单因子实验结果表明，菌株DNSl0适宜生长和降解的条件范围是：温度25～35＇12，pH值5．0～8．0，培养液盐度0．1％～2％，对阿特拉津最大耐受浓度可达1200mg／L。正交实验法进一步表明，该菌株保持较好生长及降解能力的最优方案是温度30℃，pH值7．5，培养液盐度0．5％。影响其降解能力的环境因素的主次顺序依次是：温度〉盐度〉pH值。     

Appendix

Abstract

An atrazine‐degrading bacterial isolate (M91–3) was able to utilize simazine and cyanazine as N sources for glucose‐dependent growth. The degradation of these three 5‐triazine herbicides was also investigated in binary and ternary mixtures. The organism used atrazine and simazine indiscriminately, whereas cyanazine degradation was slow and delayed until the depletion of the two other herbicides. There was no apparent effect of other commonly used herbicides on the rate of atrazine degradation by M91–3.     

Degrader density determines spatial variability of 2,6-dichlorobenzamide mineralisation in soil

The metabolite 2,6-dichlorobenzamide (BAM) is a frequent groundwater pollutant produced during degradation of the herbicide 2,6-dichlorobenzonitrile (dichlobenile). Spatial variability of BAM mineralisation is uncharacterized in surface soil, however, and factors controlling the heterogeneity remain unknown. We addressed these issues by sample-to-sample comparisons of BAM mineralisation rates and a range of soil characteristics at spatial scales ranging from meters to centimetres. For mineralisation assays nano-molar concentrations of labelled BAM were added to determine mineralisation rates under realistic conditions. We found a significant variability of BAM mineralisation which increased with decreasing spatial scale. BAM mineralisation rates were correlated to the density of BAM-degrading bacteria but not to water content, TOC, NH₄⁺, NO₃⁻, or pH. The genus Aminobacter, which contains the only BAM degraders known, was detected in MPN samples of BAM degraders by a specific PCR assay targeting the 16S rRNA gene, confirming a role of Aminobacter in BAM mineralisation.     

Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil

For phytoremediation of organic contaminants, plants have to host an efficiently degrading microflora. To assess the role of endophytes in alkane degradation, Italian ryegrass was grown in sterile soil with 0, 1 or 2% diesel and inoculated either with an alkane degrading bacterial strain originally derived from the rhizosphere of Italian ryegrass or with an endophyte. We studied plant colonization of these strains as well as the abundance and expression of alkane monooxygenase (alkB) genes in the rhizosphere, shoot and root interior. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.     

Influence of microbial and synthetic surfactant on the biodegradation of atrazine

The present study reports the effect of surfactants (rhamnolipids and triton X-100) on biodegradation of atrazine herbicide by strain A6, belonging to the genus Acinetobacter. The strain A6 was able to degrade nearly 80 % of the 250-ppm atrazine after 6 days of growth. The bacterium degraded atrazine by de-alkylation process. Bacterial cell surface hydrophobicity as well as atrazine solubility increased in the presence of surfactant. However, addition of surfactant to the mineral salt media reduced the rate and extent of atrazine degradation by decreasing the bioavailability of herbicide. On the contrary, addition of surfactant to atrazine-contaminated soil increased the rate and extent of biodegradation by increasing the bioavailability of herbicide. As compared to triton X-100, rhamnolipids were more efficient in enhancing microbial degradation of atrazine as a significant amount of atrazine was removed from the soil by rhamnolipids. Surfactants added for the purpose of hastening microbial degradation may have an unintended inhibitory effect on herbicide degradation depending upon contiguous condition, thus highlighting the fact that surfactant must be judiciously used in bioremediation of herbicides.     

Biodegradation of atrazine by Rhodococcus sp. BCH2 to N-isopropylammelide with subsequent assessment of toxicity of biodegraded metabolites

Atrazine is a persistent organic pollutant in the environment which affects not only terrestrial and aquatic biota but also human health. Since its removal from the environment is needed, atrazine biodegradation is achieved in the present study using the bacterium Rhodococcus sp. BCH2 isolated from soil, long-term treated with atrazine. The bacterium was capable of degrading about 75 % atrazine in liquid medium having pH 7 under aerobic and dark condition within 7 days. The degradation ability of the bacterium at various temperatures (20–60 °C), pH (range 3–11), carbon (glucose, fructose, sucrose, starch, lactose, and maltose), and nitrogen (ammonium molybdate, sodium nitrate, potassium nitrate, and urea) sources were studied for triumph optimum atrazine degradation. The results indicate that atrazine degradation at higher concentrations (100 ppm) was pH and temperature dependent. However, glucose and potassium nitrate were optimum carbon and nitrogen source, respectively. Atrazine biodegradation analysis was carried out by using high-performance thin-layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FTIR), and liquid chromatography quadrupole time-of-flight (LC/Q-TOF-MS) techniques. LC/Q-TOF-MS analysis revealed formation of various intermediate metabolites including hydroxyatrazine, N-isopropylammelide, deisopropylhydroxyatrazine, deethylatrazine, deisopropylatrazine, and deisopropyldeethylatrazine which was helpful to propose biochemical degradation pathway of atrazine. Furthermore, the toxicological studies of atrazine and its biodegraded metabolites were executed on earthworm Eisenia foetida as a model organism with respect to enzymatic (SOD and Catalase) antioxidant defense mechanism and lipid peroxidation studies. These results suggest innocuous degradation of atrazine by Rhodococcus sp. BCH2 in nontoxic form. Therefore the Rhodococcus sp.BCH2 could prove a valuable source for the eco-friendly biodegradation of atrazine pesticide.     

Degradation of atrazine by an acclimatized soil fungal isolate

A fungal strain able to use atrazine (2-chloro-4-ethylamino-5-isopropylamino-1,3,5-triazine) as a source of nitrogen was isolated from a corn field soil that has been previously treated with the herbicide. This strain was purified and acclimatized to atrazine at a higher level in the laboratory. A supplemented N was required to trigger the reaction. Atrazine was degraded at a faster rate in inoculated mineral salt medium (MSM) than non-inoculated MSM. Within 20 days, nearly 34% of the atrazine was degraded in inoculated medium while only 2% of the herbicide was degraded in non-inoculated medium. Degradation of atrazine by the isolated fungal strain was also studied in sterile and non-sterile soil to determine the compatibility of the isolated strain with native microorganisms in soil. The degradation of atrazine was found to be more in inoculated sterile soil than in inoculated non-sterile soil. Cell free extract (CFE) of fungal mycelium degraded about 50% of the atrazine in buffer in 96 hours compared to the control. Four atrazine metabolites were isolated and characterized by LCMS. On the basis of morphological parameters the isolate was identified as Penicillium species. Results indicated that the microorganism may be useful for remediation of atrazine-contaminated soil.     

Inoculation of an atrazine-degrading strain,Chelatobacter heintzii Cit1, in four different soils: effects of different inoculum densities

The possibility to improve atrazine degradation in soils by bioaugmentation was studied. The atrazine-mineralizing strain, Chelatobacter heintzii Cit1, was inoculated in four sterile and four non-sterile soils, at varying inoculum densities. Two soils, which had shown enhanced atrazine mineralization, were used to determine which inoculum density was capable of restoring their original mineralizing capacity after sterilization. The two other soils, with intermediate and low capacity to mineralize atrazine, were used in order to demonstrate that atrazine mineralization in such soils could be improved by inoculation. Mineralization kinetics were fitted using the Gompertz model. In the case of soils adapted to atrazine mineralization, inoculation of C. heintzii did not accelerate the rate of atrazine mineralization, which was essentially performed by the indigenous microflora. However, with soils that did not mineralize atrazine, the introduction of 10(4) cfug(-1) resulted in a 3-fold increase of atrazine mineralization capacity.     

Isolation and characterization of a Rhodococcus strain with phenol-degrading ability and its potential use for tannery effluent biotreatment

Introduction

Wastewater derived from leather production may contain phenols, which are highly toxic, and their degradation could be possible through bioremediation technologies.

Materials, methods and results

In the present work, microbial degradation of phenol was studied using a tolerant bacterial strain, named CS1, isolated from tannery sediments. This strain was able to survive in the presence of phenol at concentrations of up to 1,000?mg/L. On the basis of morphological and biochemical properties, 16S rRNA gene sequencing, and phylogenetic analysis, the isolated strain was identified as Rhodococcus sp. Phenol removal was evaluated at a lab-scale in Erlenmeyer flasks and at a bioreactor scale in a stirred tank reactor. Rhodococcus sp. CS1 was able to completely remove phenol in a range of 200 to 1,000?mg/L in mineral medium at 30 ± 2?°C and pH 7 as optimal conditions. In the stirred tank bioreactor, we studied the effect of some parameters, such as agitation (200?C600 rpm) and aeration (1?C3?vvm), on growth and phenol removal efficiency. Faster phenol biodegradation was obtained in the bioreactor than in Erlenmeyer flasks, and maximum phenol removal was achieved at 400?rpm and 1 vvm in only 12?h. Furthermore, Rhodococcus sp. CS1 strain was able to grow and completely degrade phenols from tannery effluents after 9?h of incubation.

Conclusion

Based on these results, Rhodococcus sp. CS1 could be an appropriate microorganism for bioremediation of tannery effluents or other phenol-containing wastewaters.     

Degradation of chlorpyrifos by an endophytic bacterium of the Sphingomonas genus (strain HJY) isolated from Chinese chives (Allium tuberosum)

The degradation of chlorpyrifos (CP) by an endophytic bacterial strain (HJY) isolated from Chinese chives (Allium tuberosum Rottl. ex Spreng) was investigated. Strain HJY was identified as Sphingomonas sp. based on morphological, physiological, and biochemical tests and a 16S rDNA sequence analysis. Approximately 96% of 20 mg L^?1 CP was degraded by strain HJY over 15 days in liquid minimal salts medium (MSM). The CP degradation rate could also be increased by glucose supplementation. The optimal conditions for the removal of 20 mg L^?1 CP by strain HJY in MSM were 2% inoculum density, pH 6.0, and 30–35°C. The CP degradation rate constant and half-life were 0.2136 ± 0.0063 d^?1 and 3.2451 ± 0.0975 d, respectively, under these conditions, but were raised to 0.7961 ± 0.1925 d^?1 and 0.8707 ± 0.3079 d with 1% glucose supplementation. The detection of metabolic products and screening for degrading genes indicated that O,O-diethyl O-3,5,6-trichloropyridinol was the major degradation product from CP, while it was likely that some functional genes were undetected and the mechanism responsible for CP degradation by strain HJY remained unknown. Strain HJY is potentially useful for the reduction of CP residues in Chinese chives and may be used for the in situ phytoremediation of CP.     

Biodegradation of tribenuron methyl that is mediated by microbial acidohydrolysis at cell-soil interface

Tribenuron methyl (TBM) is a member of the sulfonylurea herbicide family and is widely used in weed control. Due to its phytotoxicity to rotating-crops, concerns on TBM-pollution to soil have been raised. In this study, experimental results indicated that microbial activity played a key role in TBM removal from polluted soil. Twenty-six bacterial strains were isolated and their degradation of TBM was evaluated. Serratia sp. strain BW30 was selected and subjected to further investigation on its degradative mechanism. TBM degradation by strain BW30 was dependent on glucose that was converted into lactic or oxalic acids. HPLC-MS analysis revealed two end-products from TBM degradation, and they were identical to the products from TBM acidohydrolysis. Based on this observation, it is proposed that microbe-mediated acidohydrolysis of TBM was involved in TBM degradation in soil, and possible application of this observation in bioremediation of TBM-polluted soil is discussed.