Foliar N2 O emissions constitute a significant source to atmosphere.

Nitrous oxide (N2 O) is a potent greenhouse gas and causes stratospheric ozone depletion. While the emissions of N2 O from soil are widely recognized, recent research has shown that terrestrial plants may also emit N2 O from their leaves under controlled laboratory conditions. However, it is unclear whether foliar N2 O emissions are universal across varying plant taxa, what the global significance of foliar N2 O emissions is, and how the foliage produces N2 O in situ. Here we investigated the abilities of 25 common plant taxa, including trees, shrubs and herbs, to emit N2 O under in situ conditions. Using 15 N isotopic labeling, we demonstrated that the foliage-emitted N2 O was predominantly derived from nitrate. Moreover, by selectively injecting biocide in conjunction with the isolating and back-inoculating of endophytes, we demonstrated that the foliar N2 O emissions were driven by endophytic bacteria. The seasonal N2 O emission rates ranged from 3.2 to 9.2 ng N2 O-N g-1 dried foliage h-1 . Extrapolating these emission rates to global foliar biomass and plant N uptake, we estimated global foliar N2 O emission to be 1.21 and 1.01 Tg N2 O-N year-1 , respectively. These estimates account for 6%-7% of the current global annual N2 O emission of 17 Tg N2 O-N year-1 , indicating that in situ foliar N2 O emission is a universal process for terrestrial plants and contributes significantly to the global N2 O inventory. This finding highlights the importance of measuring foliar N2 O emissions in future studies to enable the accurate assigning of mechanisms and the development of effective mitigation.

Genome-Resolved Metagenomics and Denitrifying Strain Isolation Reveal New Insights into Microbial Denitrification in the Deep Vadose Zone.

The heavy use of nitrogen fertilizer in intensive agricultural areas often leads to nitrate accumulation in subsurface soil and nitrate contamination in groundwater, which poses a serious risk to public health. Denitrifying microorganisms in the subsoil convert nitrate to gaseous forms of nitrogen, thereby mitigating the leaching of nitrate into groundwater. Here, we investigated denitrifying microorganisms in the deep vadose zone of a typical intensive agricultural area in China through microcosm enrichment, genome-resolved metagenomic analysis, and denitrifying bacteria isolation. A total of 1000 metagenome-assembled genomes (MAGs) were reconstructed, resulting in 98 high-quality, dereplicated MAGs that contained denitrification genes. Among them, 32 MAGs could not be taxonomically classified at the genus or species level, indicating that a broader spectrum of taxonomic groups is involved in subsoil denitrification than previously recognized. A denitrifier isolate library was constructed by using a strategy combining high-throughput and conventional cultivation techniques. Assessment of the denitrification characteristics of both the MAGs and isolates demonstrated the dominance of truncated denitrification. Functional screening revealed the highest denitrification activity in two complete denitrifiers belonging to the genus Pseudomonas. These findings greatly expand the current knowledge of the composition and function of denitrifying microorganisms in subsoils. The constructed isolate library provided the first pool of subsoil-denitrifying microorganisms that could facilitate the development of microbe-based technologies for nitrate attenuation in groundwater.

Anthropogenic N input increases global warming potential by awakening the "sleeping" ancient C in deep critical zones.

Even a small net increase in soil organic carbon (SOC) mineralization will cause a substantial increase in the atmospheric CO2 concentration. It is widely recognized that the SOC mineralization within deep critical zones (2 to 12 m depth) is slower and much less influenced by anthropogenic disturbance when compared to that of surface soil. Here, we showed that 20 years of nitrogen (N) fertilization enriched a deep critical zone with nitrate, almost doubling the SOC mineralization rate. This result was supported by corresponding increases in the expressions of functional genes typical of recalcitrant SOC degradation and enzyme activities. The CO2 released and the SOC had a similar 14C age (6000 to 10,000 years before the present). Our results indicate that N fertilization of crops may enhance CO2 emissions from deep critical zones to the atmosphere through a previously disregarded mechanism. This provides another reason for markedly improving N management in fertilized agricultural soils.

Nitrate as an alternative electron acceptor destabilizes the mineral associated organic carbon in moisturized deep soil depths.

Numerous studies have investigated the effects of nitrogen (N) addition on soil organic carbon (SOC) decomposition. However, most studies have focused on the shallow top soils <0.2 m (surface soil), with a few studies also examining the deeper soil depths of 0.5-1.0 m (subsoil). Studies investigating the effects of N addition on SOC decomposition in soil >1.0 m deep (deep soil) are rare. Here, we investigated the effects and the underlying mechanisms of nitrate addition on SOC stability in soil depths deeper than 1.0 m. The results showed that nitrate addition promoted deep soil respiration if the stoichiometric mole ratio of nitrate to O2 exceeded the threshold of 6:1, at which nitrate can be used as an alternative acceptor to O2 for microbial respiration. In addition, the mole ratio of the produced CO2 to N2O was 2.57:1, which is close to the theoretical ratio of 2:1 expected when nitrate is used as an electron acceptor for microbial respiration. These results demonstrated that nitrate, as an alternative acceptor to O2, promoted microbial carbon decomposition in deep soil. Furthermore, our results showed that nitrate addition increased the abundance of SOC decomposers and the expressions of their functional genes, and concurrently decreased MAOC, and the ratio of MAOC/SOC decreased from 20% before incubation to 4% at the end of incubation. Thus, nitrate can destabilize the MAOC in deep soils by stimulating microbial utilization of MAOC. Our results imply a new mechanism on how above-ground anthropogenic N inputs affect MAOC stability in deep soil. Mitigation of nitrate leaching is expected to benefit the conservation of MAOC in deep soil depths.

Enhanced degradation of microplastics during sludge composting via microbially-driven Fenton reaction.

It has been increasingly documented that the hydroxyl radical (•OH) can promote the transformation of organic contaminants such as microplastics (MPs) in various environments. However, few studies have sought to identify an ideal strategy for accelerating in situ MPs degradation through boosting the process of •OH production in practical applications. In this work, iron-mineral-supplemented thermophilic composting (imTC) is proposed and demonstrated for enhancing in situ degradation of sludge-based MPs through strengthening •OH generation. The results show that the reduction efficiency of sludge-based MPs abundance was about 35.93% in imTC after treatment for 36 days, which was 38.99% higher than that of ordinary thermophilic composting (oTC). Further investigation on polyethylene-microplastics (PE-MPs) suggested that higher abundance of •OH (the maximum value was 408.1 µmol·kg-1) could be detected on the MPs isolated from imTC through microbially-mediated redox transformation of iron oxides, as compared to oTC. Analyses of the physicochemical properties of the composted PE-MPs indicated that increased •OH generation could largely accelerate the oxidative degradation of MPs. This work, for the first time, proposes a feasible strategy to enhance the reduction efficiency of MPs abundance during composting through the regulation of •OH production.

Enantioseparation of amino acid and mandelic acid enantiomers using Josiphos-metal complexes as chiral extractants.

The development of new and efficient chiral extractants has always been the research hotspot and difficulty in the field of chiral extraction. Josiphos, a famous ferrocene derivative catalyst, is employed as a chiral extractant in enantioseparation of amino acid and mandelic acid enantiomers. The influences of metal ions, organic solvents, pH of the aqueous solution, extractant concentrations, and extraction temperature on enantioselectivities are systematically studied. The result reveals that Josiphos-Pd has good capabilities to enantioseparate 4-nitro-phenylalanine (Nphe), 3-chloro-phenylglycine (Cpheg), and mandelic acid (MA) with separation factors (α) of 3.30, 2.65, and 2.18, respectively. The pH of the aqueous phase and Josiphos-Pd concentration affect the extraction significantly, whereas extraction temperature shows little influence. After optimizing by response surface method, the mathematical models for extractions are established. And the highest experimental performance factors (pf) for Nphe, Cpheg, and MA are 0.1843, 0.1335, and 0.08884, respectively.

Reactive Oxygen Species Promote Nitrous Oxide (N2O) Emissions from Soil/Sediment during the Anoxic-Oxic Transition.

Reactive oxygen species (ROS)-induced element/pollutant geochemical processes in fluctuating anoxic-oxic areas have received increasing attention in recent years. Nitrous oxide (N2O) is a strong greenhouse gas; however, the relationship between ROS and N2O emissions in these areas has not been established. This work revealed the essential role of ROS in promoting N2O emissions in soil/sediment during the anoxic-oxic transition. ROS decreased the rate of nitrate reduction by 26-31% and increased N2O emissions by 8.8-31.3% (at 48 h). ROS-induced N2O emission was via inhibiting the step of N2O reduction. During the anoxic-oxic transition, the contribution of ROS to inhibit the step of N2O reduction was higher than 52.6%, demonstrating the important role of ROS. The downregulated relative transcription of the NosZ gene demonstrated inhibition at the gene level. Hydrogen peroxide was the dominant ROS species inhibiting N2O reduction, while the role of hydroxyl radicals was negligible, suggesting a different behavior of N2O emission with common pollutant conversion induced by ROS during the anoxic-oxic transition. This study demonstrated an overlooked factor in promoting N2O emission in the soil/sediment and appealed to a re-examination of the mechanism of N2O emissions in fluctuating anoxic-oxic areas.

Extensive production and evolution of free radicals during composting.

The production of free radicals has been widely documented in natural systems, where they play an important role in most organic matter and contaminants transformation. Here, the production and evolution of free radicals were systematically investigated during composting. Results indicated that multiple reactive oxygen species and environmentally persistent free radicals (G-factor 2.003-2.004) were generated with dynamic changes during composting. The ·OH yield fluctuated significantly with a maximum content of 365.7-1,262.3 µmol/kg at the thermophilic phase of composting, which was closely correlated with the changes of Fe (II) (Pearson's r = 0.928-0.932) and the electron-donating capacity of humus (Pearson's r = 0.958-0.896) during composting. Further investigation suggested that microorganisms driven iron/humus redox conversion could contribute to the production and dynamic changes of free radical during composting. These findings highlight the abiotic processes involving free radicals, and provide a new perspective for humification and contaminants removal during composting.

Free radicals accelerate in situ ageing of microplastics during sludge composting.

Composting is the last "barrier" for microplastics (MPs) in the entry of organic solid wastes into the environment. The transformation of MPs is thought to be mainly driven by microorganisms during composting, whereas the contribution of abiotic processes that involve free radicals is often overlooked. Herein, we provide initial evidence for the generation of free radicals during sludge composting, including environmental persistent free radicals and reactive oxygen species, which accelerate the oxidative degradation of MPs. The ·OH yield of composting fluctuated greatly from 23.03 to 277.18 µmol/kg during composting, which was closely related to the dynamic changes in Fe(II) (R2 = 0.926). Analyses of the composted MPs physicochemical properties indicated that MPs were aged gradually with molecular weights decrease from 18% to 27% and carbonyl index value increase from 0.23 to 0.52. Further investigation suggested that the microbially-mediated redox transformation of iron oxides could occur on the MPs surface accompanied by the production of abundant free radicals, thereby leading to the damage of MPs during composting. These results reveal the critical role of free radicals in MPs ageing under oxic/anoxic alternation conditions of composting and provide new insights into the bio-chemical mechanism of contaminant removal or transformation during sludge composting.

Deep convolutional neural network for preoperative prediction of microvascular invasion and clinical outcomes in patients with HCCs.

OBJECTIVES: We aimed to develop and validate a deep convolutional neural network (DCNN) model for preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) and its clinical outcomes using contrast-enhanced computed tomography (CECT) in a large population of candidates for surgery. METHODS: This retrospective study included 1116 patients with HCC who had undergone preoperative CECT and curative hepatectomy. Radiological (R), DCNN, and combined nomograms were constructed in a training cohort (n = 892) respectively based on clinicoradiological factors, DCNN probabilities, and all factors; the performance of each model was confirmed in a validation cohort (n = 244). Accuracy and the AUC to predict MVI were calculated. Disease-free survival (DFS) and overall survival (OS) after surgery were recorded. RESULTS: The proportion of MVI-positive patients was respectively 38.8% (346/892) and 35.7 % (87/244) in the training and validation cohorts. The AUCs of the R, DCNN, and combined nomograms were respectively 0.809, 0.929, and 0.940 in the training cohorts and 0.837, 0.865, and 0.897 in the validation cohort. The combined nomogram outperformed the R nomogram in the training (p < 0.001) and validation (p = 0.009) cohorts. There was a significant difference in DFS and OS between the R, DCNN, and combined nomogram-predicted groups with and without MVI (p < 0.001). CONCLUSIONS: The combined nomogram based on preoperative CECT performs well for preoperative prediction of MVI and outcome. KEY POINTS: • A combined nomogram based on clinical information, preoperative CECT, and DCNN can predict MVI and clinical outcomes of patients with HCC. • DCNN provides added diagnostic ability to predict MVI. • The AUCs of the combined nomogram are 0.940 and 0.897 in the training and validation cohorts, respectively.

Two-Trait Predictor of Venous Invasion on Contrast-Enhanced CT as a Preoperative Predictor of Outcomes for Early-Stage Hepatocellular Carcinoma After Hepatectomy.

OBJECTIVES: This study aimed to assess the effectiveness of the two-trait predictor of venous invasion (TTPVI) on contrast-enhanced computed tomography (CECT) for the preoperative prediction of clinical outcomes in patients with early-stage hepatocellular carcinoma (HCC) after hepatectomy. METHODS: This retrospective study included 280 patients with surgically resected HCC who underwent preoperative CECT between 2012 and 2013. CT imaging features of HCC were assessed, and univariate and multivariate Cox regression analyses were used to evaluate the CT features associated with disease-free survival (DFS) and overall survival (OS). Subgroup analyses were used to summarized the hazard ratios (HRs) between patients in whom TTPVI was present and those in whom TTPVI was absent using a forest plot. RESULTS: Capsule appearance [HR, 0.504; 95% confidence interval (CI), 0.341-0.745; p < 0.001], TTPVI (HR, 1.842; 95% CI, 1.319-2.572; p < 0.001) and high level of alanine aminotransferase (HR, 1.620; 95% CI, 1.180-2.225, p = 0.003) were independent risk factors for DFS, and TTPVI (HR, 2.509; 95% CI, 1.518-4.147; p < 0.001), high level of alpha-fetoprotein (HR, 1.722; 95% CI, 1.067-2.788; p = 0.026), and gamma-glutamyl transpeptidase (HR, 1.787; 95% CI, 1.134-2.814; p = 0.026) were independent risk factors for OS. A forest plot revealed that the TTPVI present group had lower DFS and OS rates in most subgroups. Patients in whom TTPVI was present in stages I and II had a lower DFS and OS than those in whom TTPVI was absent. Moreover, there were significant differences in DFS (p < 0.001) and OS (p < 0.001) between patients classified as Barcelona Clinic Liver Cancer stage A in whom TTPVI was absent and in whom TTPVI was present. CONCLUSIONS: TTPVI may be used as a preoperative biomarker for predicting postoperative outcomes for patients with early-stage HCC.

An improved microelectrode method reveals significant emission of nitrous oxide from the rhizosphere of a long-term fertilized soil in the North China Plain.

Microsensors are able to accurately quantify nitrous oxide (N2O) emissions in microenvironments at high spatio-temporal resolution; yet, limited studies have been conducted on agricultural soils due to the inability to obtain electrical signal under conditions of low soil moisture. This study improved the calibration of a microelectrode for measuring N2O emissions from agricultural soil. The microelectrode was applied to evaluate the effect of long-term fertilization with mineral fertilizer (NPK), complemented with pig manure (MNPK), straw (SNPK), or without fertilizer (CK), all with and without urea addition, on N2O emissions from the soil, with explicit separation of the rhizosphere and the bulk soil compartments. The use of soil solution instead of pure water for calibration of the microelectrode doubled the signal and significantly improved the sensor sensitivity. The optimal electrolytic concentration of the soil solution, expressed as water: soil ratio, was found at the maximum vertex of the quadratic equation fitted on the slope values of the calibration equations for different soil solutions. The application of the calibrated microelectrode revealed significantly higher N2O emission from the rhizosphere compared to the bulk soil, accounting for 60% of the total emission. For the bulk soil, MNPK significantly increased N2O emissions compared to SNPK and NPK, whereas the differences between these treatments for the rhizosphere soil were insignificant. The statistical modeling revealed significant relation of the N2O emission with soil inorganic nitrogen contents and an additive effect of treatment (MNPK and SNPK), urea addition and rhizosphere soil. This study provides novel insights into the use of microelectrodes for measuring N2O emissions from the soil microenvironment and also points on the rhizosphere compartment and the management practices of agroecosystems able to reduce the N2O emission from agriculture.

Dissecting the Structural and Conductive Functions of Nanowires in Geobacter sulfurreducens Electroactive Biofilms.

Conductive nanowires are thought to contribute to long-range electron transfer (LET) in Geobacter sulfurreducens anode biofilms. Three types of nanowires have been identified: pili, OmcS, and OmcZ. Previous studies highlighted their conductive function in anode biofilms, yet a structural function also has to be considered. We present here a comprehensive analysis of the function of nanowires in LET by inhibiting the expression of each nanowire. Meanwhile, flagella with poor conductivity were expressed to recover the structural function but not the conductive function of nanowires in the corresponding nanowire mutant strain. The results demonstrated that pili played a structural but not a conductive function in supporting biofilm formation. In contrast, the OmcS nanowire played a conductive but not a structural function in facilitating electron transfer in the biofilm. The OmcZ nanowire played both a structural and a conductive function to contribute to current generation. Expression of the poorly conductive flagellum was shown to enhance biofilm formation, subsequently increasing current generation. These data support a model in which multiheme cytochromes facilitate long-distance electron transfer in G. sulfurreducens biofilms. Our findings also suggest that the formation of a thicker biofilm, which contributed to a higher current generation by G. sulfurreducens, was confined by the biofilm formation deficiency, and this has applications in microbial electrochemical systems. IMPORTANCE The low power generation of microbial fuel cells limits their utility. Many factors can affect power generation, including inefficient electron transfer in the anode biofilm. Thus, understanding the mechanism(s) of electron transfer provides a pathway for increasing the power density of microbial fuel cells. Geobacter sulfurreducens was shown to form a thick biofilm on the anode. Cells far away from the anode reduce the anode through long-range electron transfer. Based on their conductive properties, three types of nanowires have been hypothesized to directly facilitate long-range electron transfer: pili, OmcS, and OmcZ nanowires. However, their structural contributions to electron transfer in anode biofilm have not been elucidated. Based on studies of mutants lacking one or more of these facilitators, our results support a cytochrome-mediated electron transfer process in Geobacter biofilms and highlight the structural contribution of nanowires in anode biofilm formation, which contributes to biofilm formation and current generation, thereby providing a strategy to increase current generation.

Electrodes Donate Electrons for Nitrate Reduction in a Soil Matrix via DNRA and Denitrification.

Microbial strains and indigenous microbiota in soil slurries have been reported to use electrons from electrodes for nitrate (NO3-) reduction. However, few studies have confirmed this in a soil matrix hitherto. This study investigated if, and how, an electric potential affected NO3- reduction in a soil matrix. The results showed that, compared to a control treatment, applying an electric potential of -0.5 V versus the standard hydrogen electrode (SHE) significantly increased the relative abundance of NO3--reducing microbes (e.g., Alcaligenaceae and Pseudomonadaceae) and the abundances of the nrfA, nirK, nirS, and nosZ genes in soil matrices. Meanwhile, the electric potential treatment doubled the NO3- reduction rate and significantly increased the rates of production of ammonium (NH4+), dinitrogen (N2), and nitrous oxide (N2O). The amount of NO3--N reduced under the electric potential treatment was comparable to the sum of the amounts of N observed in the increased N2O, N2, NH4+, and nitrite (NO2-) pools. An open-air experiment showed that the electric potential treatment promoted soil NO3- reduction with a spatial scale of at least 38 cm. These results demonstrated that an electric potential treatment could enhance NO3- reduction via both denitrification and dissimilatory NO3- reduction to ammonium (DNRA) in the soil matrix. The mechanisms revealed in this study have implications for the future development of potential techniques for enhancing NO3- reduction in the vadose zone and consequently reducing the risk of NO3- leaching.

Organic carbon availability limiting microbial denitrification in the deep vadose zone.

Microbes in the deep vadose zone play an essential role in the mitigation of nitrate leaching; however, limited information is available on the mechanisms of microbial denitrification due to sampling difficulties. We experimentally studied the factors that affect denitrification in soils collected down to 10.5 meters deep along the soil profile. After an anoxic pre-incubation, denitrification rates moderately increased and the N2 O/(N2 O + N2 ) ratios declined while the microbial abundance and diversity did not change significantly in most of the layers. Denitrification rate was significantly enhanced and the abundance of the denitrification genes was simultaneously elevated by the increased availability of organic carbon in all studied layers, to a greater extent in the subsurface layers than in the surface layers, suggesting the severe scarcity of carbon in the deep vadose zone. The genera Pseudomonas and Bacillus, which are made up of a number of species that have been previously identified as denitrifiers in soil, were the major taxa that respond to carbon addition. Overall, our results suggested that the limited denitrification in the deep vadose zone is not because of the lack of denitrifiers, but due to the low abundance of denitrifiers which is caused by low carbon availability.

Perturbation-free measurement of in situ di-nitrogen emissions from denitrification in nitrate-rich aquatic ecosystems.

Increased production of reactive nitrogen (Nr) from atmospheric di-nitrogen (N2) has greatly contributed to increased food production. However, enriching the biosphere with Nr has also caused a series of negative effects on global ecosystems, especially aquatic ecosystems. The main pathway converting Nr back into the atmospheric N2 pool is the last step in the denitrification process. Despite several attempts, there is still a need for perturbation-free methods for measuring in situ N2 fluxes from denitrification in aquatic ecosystems at the field scale. Such a method is needed to comprehensively quantify the N2 fluxes from aquatic ecosystems. Here we observed linear relationships between the δ15N-N2O signatures and the logarithmically transformed N2O/(N2+N2O) emission ratios. Through independent measurements, we verified that the perturbation-free N2 flux from denitrification in nitrate-rich aquatic ecosystems can be inferred from these linear relationships. Our method allowed the determination of field-scale in situ N2 fluxes from nitrate-rich aquatic ecosystems both with and without overlaying water. The perturbation-free in situ N2 fluxes observed by the new method were almost one order of magnitude higher than those by the sediment core method. The ability of aquatic ecosystems to remove Nr may previously have been severely underestimated.

Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml(-1)], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80%) of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

Binding of pyrene to dissolved organic matters: fractionation and characterization.

Based on a modified Leenheer DOM fractionation scheme, fractionation of DOM from the paddy soil was conducted by using XAD-8 resin into hydrophobic bases (HOB), hydrophobic acids(HOA), acid-insoluble matter (AIM), hydrophobic neutrals (HON) and hydrophilic matter (HIM). In total carbon content of DOM, 35.32% were the HIM and only 0.73% the HOB. However, HOA and AIM altogether occupied 53.45%, while the HON fraction represented 10%. The sorption experiments were conducted to determine the sorption capacity of pyrene on unfractionated DOM and its fractions. Elemental analysis, 1H-NMR and FTIR spectra were carried out on unfractionated DOM and its fractions to examine the relationship between the structure of DOM and partition coefficients (K(oc)). The results showed that HON had a greater affinity for binding pyrene than other fractions. While HON was characterized by large long-chain alkylate (aliphatic structure). AIM exhibited relative higher K(oc) values than HOA and HIM, due to much aromatic structure in AIM, while the high content of carboxylic groups of HOA and HIM depressed their binding capacity. This study demonstrated HON is a key subcomponents of DOM in binding of pyrene, in other words, aliphalic structure in DOM play an important role in binding of pyrene.