Nitrate removal by Thiobacillus denitrificans immobilized on poly(vinyl alcohol) carriers

Nitrate contamination is becoming a widespread environmental problem, and autotrophic denitrification with Thiobacillus denitrificans is a promising process considering efficiency, cost and maintenance. The denitrification efficiencies of T. denitrificans were compared in batch reactors between free cells and cells immobilized on polyvinyl alcohol (PVA) carriers made with thrice freezing/thawing and boric acid methods. The results indicated that the free cell reactor of T. denitrificans added with 10% (v/v) of PVA carrier made by thrice freezing/thawing (PVA-TFT) exhibited faster in S(2)O(3)(2-)-S consumption, SO(4)(2-) generation, and NO(3)(-)-N denitrification, with corresponding values being 165 mg (S(2)O(3)(2-)-S)/L.d, 491 mg (SO(4)(2-))/Ld, and 44 mg (NO(3)(-)-N)/Ld, which were increased by 50%, 61%, and 57% respectively compared to the control reactor with only free cells. Inhibition of denitrification by accumulated SO(4)(2-) in PVA-TFT reactor appeared at the concentration of approximately 6000 mg (SO(4)(2-))/L, and 75% of NO(3)(-)-N removal efficiency was achieved after 12d operation under the condition of initial 700 mg/L NO(3)(-)-N concentration.     

Chemical reduction of an unbuffered nitrate solution using catalyzed and uncatalyzed nanoscale iron particles

Uncatalyzed and catalyzed nanoscale Fe(0) systems were employed for the denitrification of unbuffered 40 mgN L(-1) nitrate solutions at initial neutral pH. Compared to microscale Fe(0) (<100 mesh), the efficiency and rate of nitrate removal using uncatalyzed and catalyzed nano-Fe(0) were highly promoted, in which the maximum promoted rate was obtained using copper-catalyzed nano-Fe(0) (nano-Cu/Fe). Nitrate first-order degradation rate constants (k(obs)) decreased significantly (>70%) with aged nano-Fe(0) and aged nano-Cu/Fe, and were recovered with NaBH(4) as reductants at levels of about 85 and 75%, respectively. Activation energies (E(a)) of nitrate reduction over the temperature range of 10-60 degrees C were 42.5 kJ mol(-1) for microscale Fe(0), 25.8 kJ mol(-1) for nano-Fe(0) and 16.8 kJ mol(-1) for nano-Cu/Fe. Unlike microscale Fe(0), the kinetics of denitrification by nano-Fe(0) and nano-Cu/Fe began to show characteristics of mass transport in addition to chemical reaction control. Ammonium was the predominant end product in all the systems. However, as for nitrite, 40% of the degraded nitrate persisted in the nano-Cu/Fe system. Thus, relative to nano-Cu/Fe, nano-Fe(0) is a potential reductant for denitrification of groundwater as far as toxic nitrite generation is concern.     

Nitrate reduction over nanoscale zero-valent iron prepared by hydrogen reduction of goethite

Nitrate reduction with nanoscale zero-valent iron (NZVI) was reported as a potential technology to remove nitrate from nitrate-contaminated water. In this paper, nitrate reduction with NZVI prepared by hydrogen reduction of natural goethite (NZVI-N, -N represents natural goethite) and hydrothermal goethite (NZVI-H, -H represents hydrothermal goethite) was conducted. Besides, the effects of reaction time, nitrate concentration, iron-to-nitrate ratio on nitrate removal rate over NZVI-H and NZVI-N were investigated. To prove their excellent nitrate reduction capacities, NZVI-N and NZVI-H were compared with ordinary zero-valent iron (OZVI-N) through the static experiments. Based on all above investigations, the mechanism of nitrate reduction with NZVI-N was proposed. The result showed that reaction time, nitrate concentration, iron-to-nitrate ratio played an important role in nitrate reduction by NZVI-N and NZVI-H. Compared with OZVI, NZVI-N and NZVI-H showed little relationship with pH. And NZVI-N for nitrate composition offers a higher stability than NZVI-H because of the existence of Al-substitution. Furthermore, NZVI-N, prepared by hydrogen reduction of goethite, has higher activity for nitrate reduction and the products contain hydrogen, nitrogen, NH₄⁺, a little nitrite, but no NOx, meanwhile NZVI-N was oxidized to Fe²⁺. It is a relatively easy and cost-effective method for nitrate removal, so NZVI-N reducing nitrate has a great potential application in nitrate removal of groundwater.     

Influence of alkalinity on the stabilization of municipal solid waste in anaerobic simulated bioreactor

Four simulated landfill anaerobic bioreactors were performed to investigate the influence of alkalinity on the anaerobic treatment of municipal solid waste (MSW). Leachate was recirculated in all the four reactors. One reactor was operated without alkalinization. The other three were operated under alkaline conditions. Na(2)CO(3), NaHCO(3) and NaOH were added to leachate in the second, third and fourth reactor, respectively. Experimental results showed that CO(3)(2-) and HCO(3)(-) addition had a more pronounced effect on MSW stabilization while the effect of addition of OH(-) was weak. The concentration of COD, BOD(5), total nitrogen (TN), ammonium nitrogen (NH(4)(+)-N) and nitrate nitrogen (NO(3)(-)-N), etc. in leachate significantly reduced in four reactors. The removal efficiencies were 90.56%, 92.21%, 92.74% and 90.29% for COD, 66.45%, 72.38%, 68.62% and 68.44% for NO(3)(-)-N, and 96.5%, 98.75%, 97.75% and 98% for NO(2)(-)-N in the control, Na(2)CO(3), NaHCO(3) and OH(-) added reactors, respectively. The final BOD(5)/COD was 0.262, 0.104, 0.124, and 0.143, and pH was 7.13, 7.28, 7.42, and 7.24 for control, Na(2)CO(3) added, NaHCO(3) added, and OH(-) added reactor, respectively. Therefore, alkalinity addition had positive effect on the stabilization of MSW.     

Catalytic wet peroxide oxidation of azo dye (Direct Blue 15) using solvothermally synthesized copper hydroxide nitrate as catalyst

Copper hydroxide nitrate (Cu(2)(OH)(3)NO(3)) was synthesized solvothermally in anhydrous ethanol and characterized by XRD, FTIR, TG-DTA and SEM. The peroxide degradation of an azo dye (Direct Blue 15) on this material was evaluated by examining catalyst loading, initial pH, hydrogen peroxide dosage, initial dye concentration and temperature. The leaching of Cu from the copper hydroxide nitrate during the reaction was also measured. The copper hydroxide nitrate synthesized solvothermally, which was of a novel spherical morphology with complex secondary structures and contained high-dispersed Cu(2)O impurity, showed good performance for oxidation degradation of the azo dye, especially high catalytic activity, high utilization of hydrogen peroxide and a wide pH range, whereas the copper hydroxide nitrate synthesized by the direct reaction of copper nitrate and sodium hydroxide showed low catalytic activity.     

Magnetic gamma-Fe(2)O(3) nanoparticles coated with poly-l-cysteine for chelation of As(III), Cu(II), Cd(II), Ni(II), Pb(II) and Zn(II)

An anaerobic attached-growth bioreactor (AAGBR) of 3.52 L was operated for 510 days to treat sulfide-laden organic wastewater where nitrate and nitrite were introduced as electron acceptors. When the influent sulfide was kept at 200mg S(2-)-S/L and organic carbon was increased from 20 to 33.6 mg C/L, and the hydraulic retention time decreased from 41.4 to 2.67 h, the removal rates of sulfide and organic carbon reached 99.9% and 91.8% at the loading rates of 1800 mg S(2-)-S/(Ld) and 302.4 mg C/(Ld), respectively. Simultaneously, the introduced electron acceptors of nitrate and nitrite were, respectively, removed by 99.9% and 99.9% at the loading rates of 472.5 mg NO(3)(-)-N/(Ld) and 180 mg NO(2)(-)-N/(Ld). Inside the AAGBR, both autotrophic and heterotrophic denitrification processes were noted to take place. When the influent organic carbon was increased from 20 to 33.6 mg C/L, the nitrate and nitrite consumed for heterotrophic denitrification accounted for 27.3% and 48.5%, respectively. This simultaneous autotrophic and heterotrophic desulfurization-denitrification process has provided a demonstration of the possibility to eliminate sulfide and organic carbon with the presence of nitrate and nitrite.     

Effects of anion species and concentration on the removal of Cr(VI) by a microalgal isolate, Chlorella miniata

The presence of nitrate, chloride and sulfate anions on the removal of Cr(VI) by Chlorella miniata was investigated. Results of kinetic studies indicated that the equilibrium time in each anion system increased with increases of the initial salt concentration, and the inhibitory order was NO(3)(-)>Cl(-)>SO(4)(2-) for Cr(VI) removal and was SO(4)(2-)>Cl(-) approximately NO(3)(-) for the biosorption of the bioreduced Cr(III). The inhibitory effect caused by different anions was attributed to biosorption mechanism and metal speciation. Since both biosorption and bioreduction were involved in Cr(VI) removal, the presence of anions could compete with Cr(VI) for the adsorption sites, and the affinity of anions to the algal biomass followed the order of NO(3)(-)>Cl(-)>SO(4)(2-), which was consistent with their inhibitory order on Cr(VI) removal. Speciation results also indicated that the formation of CrO(3)SO(4)(2-) in the sulfate system made it easier to be adsorbed on the biomass than HCrO(4)(-). The biosorption-bioreduction model further suggested that the bioreduction rate constant k decreased with increases of anion concentrations ranging from 0 to 0.5M, and followed the order of SO(4)(2-)>Cl(-)>NO(3)(-). The biosorption constant b also decreased with anion concentrations in the range of 0-0.2M, suggesting that this parameter was more sensitive to anion effects than the k values. The higher b values in the sulfate than in the nitrate and chloride systems indicated that Cr(VI) in the sulfate system was more easily adsorbed on the algal biomass. These findings demonstrated that the presence of anions significantly affected the removal of Cr(VI) by C. miniata. Since chloride, nitrate and sulfate ions are commonly found in industrial wastewater, it may be necessary to eliminate these ions prior to chromium removal.     

Factors influencing the removal of fluoride from aqueous solution by calcined Mg-Al-CO3 layered double hydroxides

Layered double hydroxides (LDH) calcined at different temperatures (denoted as CLDH) have been demonstrated to recover their original layered structure in the presence of appropriate anions. In the light of this so-called "memory effect", a study of removal of fluoride from aqueous solution by calcined Mg-Al-CO(3)-LDH has been carried out. The LDH calcined at 500 degrees C had the highest capacity of removal of fluoride ion, because of retention of its intrinsic structure. The CLDH with an Mg/Al ratio of 2 has a remarkable ability to adsorb anions. The adsorption loading is higher for the calcined Mg-Al-LDH than for calcined Zn-Al and Ni-Al-LDH. The influence of varying the conditions for removal of fluoride, such as the pH of aqueous solution, the initial fluoride concentration, the dosage of adsorbent, and temperature on removal of fluoride have been investigated. The influence of co-existing anions in fluoride aqueous solution indicates that the percentage of removal of fluoride increased in order PO(4)(3-) < Cl(-) approximately SO(4)(2-) < Br(-) < NO(3)(-). It was found that maximum removal of fluoride from aqueous solutions was obtained in 6h at pH 6.0 with an initial concentration of 50 mg/L, and that the retention of fluoride ions by the CLDH material was 98% or higher. The residual fluoride concentration was found to be 0.4 mg/L with an initial concentration of 20 mg/L, which meets the national standard for drinking water quality. The Freundlich isotherm and Langmuir isotherm were used to fit the data of equilibrium experiments. The results of X-ray diffraction, FT-IR and TG-MS demonstrate that the adsorption phenomenon is accompanied by rehydration with concomitant uptake of fluoride ions to rebuild the initial layered structure.     

Radiation-induced reduction of diuron by gamma-ray irradiation

Diuron degradation efficiencies and the proposed mechanism by gamma-ray irradiation were investigated. Several factors that might affect the degradation values were further examined. The UV absorbances at 200-400 nm and diuron concentration decreased with the increase of radiation dose. When diuron initial concentration was 18.5 mg L(-1) and 1.0 kGy was selected as the radiation dose, diuron removal value and loss of total organic carbon were 100 and 34.1%, respectively. However, the concentration of Cl- ion increased with the increase of radiation dose. The process could be depicted by first order reaction kinetics and the reaction was mainly caused by the reaction of diuron with .OH and eaq-. The degradation efficiency decreased with the increase of initial concentration at the same radiation dose. H2O2, HCO3-, NO3-, NO2-, CH3OH and humic acid as additives reduced the degradation efficiency. Furthermore, the increase of NO3-, NO2-, CH3OH and humic acid would result in the decrease of the degradation values. The pH value could affect the removal efficiency and the degradation process was enhanced in acid condition. The pH value became lower with increasing radiation dose after gamma-ray irradiation.     

Selective inhibition of nitrite oxidation by chlorate dosing in aerobic granules

Partial nitrification was successfully achieved with addition of 5mM KClO(3) in the aerobic granules system. Batch tests demonstrated that KClO(3) selectively inhibited nitrite-oxidizing bacteria (NOB) but not ammonia-oxidizing bacteria (AOB). During stable partial nitrification, the influent pH was kept at 7.8-8.2, while the DO and temperature were not controlled in the SBR. When the NH(4)-N and COD levels were kept at 100mg/l and 400mg/l in the influent, the NH(4)-N and COD removal efficiencies reached 98.93% and 78.65%, respectively. The NO(2)-N accounted for 92.95% of the NO(χ)-N (NO(2)-N+NO(3)-N) in the effluent. Furthermore, about 90% of the chlorate was reduced to nontoxic chloride, thus it would not cause environmental problem. SEM showed that the main composition of the aerobic granules was bacilli and coccus bacteria. FISH analysis revealed that AOB became the dominant nitrifying bacteria, whereas NOB were detected only in low abundance. Chlorate could be used to control the development and maintenance of aerobic granules sludge for partial nitrification.     

Characteristics and mechanisms of phosphate adsorption onto basic oxygen furnace slag

The adsorption characteristics of phosphate adsorption on the basic oxygen furnace (BOF) slag were identified as a function of pH and ion strengths in solution. In addition, adsorption mechanisms were investigated by conducting batch tests on both the hydrolysis and phosphate adsorption process of the BOF slag, and making a comparative analysis to gain newer insights into understanding the adsorption process. Results show that the adsorption capacity from 4.97 to 3.71 mgP/g slag when the solution pH was increased from 2.0 to 13.0 and phosphate initial concentration was 50 mg/L, indicating that adsorption capacity is largely dependent upon the pH of the system. The results of the competitive adsorption between phosphate and typical anions found in wastewater, such as NO(3)(-), SO(4)(2-) and Cl(-), onto BOF slag reveal that BOF slag can selectively adsorb phosphate ions. The insignificant effect of NO(3)(-), SO(4)(2-) and Cl(-) on phosphate adsorption capacity indicates that phosphate adsorption is through a kind of inner-sphere complex reaction. During the adsorption process, the decrease of phosphate concentration in solution accompanied with an increase in pH values and concentrations of NO(3)(-), SO(4)(2-) and Cl(-) suggests that phosphate replaced the functional groups from the surface of BOF slag which infers that ligand exchange is the dominating mechanism for phosphate removal. At the same time, the simultaneous decreases in PO(4)(3-) and total calcium, magnesium and aluminum concentration in solution indicate that chemical reaction and precipitation are other mechanisms of phosphate removal.     

Chemical oxidation of methylene blue using a Fenton-like reaction

Oxidation by Fenton-like reactions is proven and economically feasible process for destruction of a variety of hazardous pollutants in wastewater. We report herein the oxidation of methylene blue, a basic dye of thiazine series using a Fenton-like reaction at normal laboratory temperature and at atmospheric pressure. The effects of different parameters like the initial concentrations of dye, Fe2+, and H2O2, pH of the solution, reaction temperature, and added electrolytes on the oxidation of the dye present in dilute aqueous solution in the concentration range (3.13-9.39)x10(-5)mol dm(-3) (10-30 mg l(-1)) have been assessed. The results indicate that the dye can be most effectively oxidized in aqueous solution at dye:Fe(2+):H2O2 molar ratio of 1:1.15:14.1. More than 98% removal of the dye could be achieved in 1h in the pH range 2.2-2.6 at 299 K which corresponds to about 81% reduction of the initial COD. The results will be useful for designing the treatment systems of various dye-containing wastewaters.     

Preparation, characterization and mechanical performance of dense β-TCP ceramics with/without magnesium substitution

Beta-tricalcium phosphate (beta-TCP) powder was prepared by a two-step process: wet precipitation of apatitic tricalcium phosphate [Ca(9)(HPO(4))(PO(4))(5)(OH)] (beta-TCP 'precursor') and calcination of the precursor at 800 degrees C for 3 h to produce beta-TCP. Magnesium-substituted tricalcium phosphate (beta-TCMP) was produced by adding Mg(NO(3))(2) . 6H(2)O into Ca(NO(3))(2) solution as Mg(2+) source before the precipitation step. The transition temperature from beta-TCP to alpha-TCP increases with the increase of Mg(2+) content in beta-TCMP. beta-TCMP with 3 mol.% Mg(2+) has beta-TCP to alpha-TCP transition temperature above 1,300 degrees C. Dense beta-TCMP (3 mol.% Mg(2+)) ceramics ( approximately 99.4% relative density) were produced by pressing the green bodies at 100 MPa and further sintering at 1,250 degrees C for 2 h. The average compressive strength of dense beta-TCP ceramics sintered at 1,100 degrees C is approximately 540 MPa, while that of beta-TCMP (3 mol.% Mg(2+)) ceramics is approximately 430 MPa.     

Negative chemical ionization GC/MS determination of nitrite and nitrate in seawater using exact matching double spike isotope dilution and derivatization with triethyloxonium tetrafluoroborate

The alkylation of nitrite and nitrate by triethyloxonium tetrafluoroborate allows determination of their ethyl esters by headspace gas chromatography/mass spectrometry (GC/MS). In the present study, significant improvement in analytical performance is achieved using negative chemical ionization providing detection limits of 150 ng/L for NO(2)(-) and 600 ng/L for NO(3)(-), an order of magnitude better than those achieved using electron impact ionization. The derivatization procedure was optimized and alkaline conditions adopted to minimize conversion of nitrite to nitrate (determined to be 0.07% at 100 mg/L NO(2)(-)) and to avoid the exchange of oxygen between the analytes and the solvent (water). Quantitation entails use of isotopically enriched standards (N(18)O(2)(-) and (15)NO(3)(-)), which also permits monitoring of potential conversion from nitrite to nitrate during the analysis (double spike isotope dilution).     

Characterization of Cr(VI) removal from aqueous solutions by a surplus agricultural waste--rice straw

The removal of Cr(VI) from aqueous solution by rice straw, a surplus agricultural byproduct was investigated. The optimal pH was 2.0 and Cr(VI) removal rate increased with decreased Cr(VI) concentration and with increased temperature. Decrease in straw particle size led to an increase in Cr(VI) removal. Equilibrium was achieved in about 48 h under standard conditions, and Cr(III), which appeared in the solution and remained stable thereafter, indicating that both reduction and adsorption played a part in the Cr(VI) removal. The increase of the solution pH suggested that protons were needed for the Cr(VI) removal. A relatively high level of NO(3)(-) notably restrained the reduction of Cr(VI) to Cr(III), while high level of SO(4)(2-) supported it. The promotion of the tartaric acid modified rice straw (TARS) and the slight inhibition of the esterified rice straw (ERS) on Cr(VI) removal indicated that carboxyl groups present on the biomass played an important role in chromium remediation even though were not fully responsible for it. Isotherm tests showed that equilibrium sorption data were better represented by Langmuir model and the sorption capacity of rice straw was found to be 3.15 mg/g.     

Biological denitrification of drinking water in a slow sand filter

Biological removal of nitrate from drinking water was studied in a slow sand filter. Optimum carbon to nitrogen ratio (C/N) was found to be 1.8 when using acetic acid in batch tests. The filtration rates impact on NO(3)-N removal through the sand filter was assessed for 22.6 mgNO(3)-N/l concentrations while keeping C/N ratio as 1.8 for acetic acid. The filtration rates varied from 0.015, 0.02, 0.03, 0.04, 0.05, and 0.06 m/h, respectively, corresponding to an overall average NO(3)-N removal efficiency of 94%. Although increasing filtration rates decreased NO(3)-N removal, effluent NO(3)-N concentrations at the effluent port were lower than the limit value. The slow sand filter process was unable to provide NO(3)-N removal rate more than 27.1 gN/(m(2)day) (0.05 m/h flow rate). The NO(3)-N removal efficiency slightly dropped from 99% to 94% when the loading rate increased from 27.1 to 32.5 g/(m(2)day), but the effluent water contained higher concentration of NO(2)-N than the standard value.     

Inoculation of a submerged filter for biological denitrification of nitrate polluted groundwater: a comparative study

Activated sludge from a wastewater treatment plant and pure culture of Hydrogenophaga pseudoflava were utilized for the development of a denitrifying biofilm in a submerged filter in order to remove nitrate from polluted groundwater. Nitrate removal efficiency, nitrite accumulation, turbidity, COD and faecal indicators persistence in the treated water were determined at different superficial hydraulic loading (10, 20 and 30 m(3)/m(2) d) and superficial nitrate loading rates (1, 2, 3, 6 and 9 Kg NO(3)(-)/m(2) d) in the submerged filter. The application of H. pseudoflava as inocula allowed better results in terms of system stability, higher superficial hydraulic loading and superficial nitrate loading rates (30 m(3)/m(2)d and 9 kg NO(3)(-) /m(2) d, respectively). These values improve those obtained when the system was inoculated with activated sludge. In addition, the pure microbial inocula improved design parameters and running of the process due to its biofilm homogeneity, obtaining treated water with better characteristics to its final use as drinking water than that obtained with an activated sludge inocula.     

Anaerobic ammonia removal in presence of organic matter: a novel route

This study describes the feasibility of anaerobic ammonia removal process in presence of organic matter. Different sources of biomass collected from diverse eco-systems containing ammonia and organic matter (OM) were screened for potential anaerobic ammonia removal. Sequential batch studies confirmed the possibility of anaerobic ammonia removal in presence of OM, but ammonia was oxidized anoxically to nitrate (at oxidation reduction potential; ORP=-248+/-25 mV) by an unknown mechanism unlike in the reported anammox process. The oxygen required for oxidation of ammonia might have been generated through catalase enzymatic activity of facultative anaerobes in mixed culture. The oxygen generation possibility by catalase enzyme route was demonstrated. Among the inorganic electron acceptors (NO(2)(-), NO(3)(-) and SO(4)(2-)) studied, NO(2)(-) was found to be most effective in total nitrogen removal. Denitrification by the developed culture was much effective and faster compared to ammonia oxidation. The results of this study show that anaerobic ammonia removal is feasible in presence of OM. The novel nitrogen removal route is hypothesized as enzymatic anoxic oxidation of NH(4)(+) to NO(3)(-), followed by denitrification via autotrophic and/or heterotrophic routes. The results of batch study were confirmed in continuous reactor operation.     

Adsorption of fluoride by waste iron oxide: the effects of solution pH, major coexisting anions, and adsorbent calcination temperature

In this study, a waste iron oxide material (BT3), which is a by-product of the fluidized-bed Fenton reaction (FBR-Fenton), was thermally treated between 200 and 900°C and was used as an efficient adsorbent for the removal of fluoride ions in an aqueous system. The highest fluoride adsorption capacity occurred at the termination of the BT3 goethite dehydroxylation phase at about 300°C calcination where both the volume of nanopores formed by dehydroxylation and the specific surface area reached their maximum values. Above 300°C, BT3 transformed to the hematite phase in which fluoride adsorption capacity decreased as calcination temperature increased. On the other hand, the effect of pH on the fluoride adsorption capacity of BT3 for various initial fluoride concentrations was examined. The optimum pH value was found to be about 4. After that efficiency decreased as pH became more alkaline. Finally, coexisting anions affected the fluoride adsorption capacity of BT3 at pH 3.9±0.2 in this order: PO(4)(3-)>SO(4)(2-)>Cl(-)>NO(3)(-).     

The removal of nitrate by nanoscale iron particles produced using the sodium borohydride method

This study was conducted to investigate removal of nitrate by nanoscale zero-valent iron (ZVI) particles in aqueous solution. ZVI particles was produced from wasted acid that is by-products of a pickling line at a steel work. The reaction activity of ZVI particles was evaluated through decomposition experiments of NO3-N aqueous solution. Addition of a larger amount of ZVI particles resulted in a higher decomposition rate. ZVI particles showed higher decomposition efficiencies than commercially purchased ZVI particles at all pH values. Both ZVIs showed a higher decomposition rate at a lower pH. Virtually no decomposition reaction was observed at pH of 4 or higher for purchased ZVI. The ZVI particles produced directly from wasted acid by the sodium borohydride method were not easy to handle because they were very small (10-200 nm) and were oxidized easily in the air.