Substrate Solution Research Articles

Catalytic Reductive Alkylation of Amines in Batch and Microflow Conditions Using a Silicon-Wafer-Based Palladium Nanocatalyst.

We describe the development of the catalytic reductive alkylation of amines with aldehydes under the atmospheric pressure of H2 using a brush-like silicon-nanostructure-supported palladium nanoparticle composite (SiNS–Pd) as a silicon-wafer-based reusable heterogeneous catalyst. The present reaction of primary and secondary amines with various aliphatic and aromatic aldehydes in the presence of the catalyst (0.02–0.05 mol % Pd) gave the corresponding secondary and tertiary amines including Lomerizine and Aticaprant in a 68% quantitative yield without overalkylation. We also designed and fabricated a flow device equipped with SiNS–Pd for microflow reactions, which was applied to the gas–liquid–solid triphasic reaction system (i.e., H2 gas, a substrate solution, and a solid catalyst). A multigram-scale reaction of aniline and benzaldehyde was demonstrated to obtain N-benzylaniline (ca. 4 g/day), in which the internal volume of the flow channel was 43 μL, the residence time was approximately 1 s, and the turnover number (TON) reached 4.0 × 104 in a continuous 24 h run (1.7 × 103 h–1; 0.50 s–1).

Storage Procedures Affect pH, Electrical Conductivity, and Nutrient Concentrations of Pour-through Leachate from Pine Bark and Peat-based Substrates

The pour-through (PT) method is used in greenhouse and nursery production to monitor nutrient availability in soilless substrates. Efficacy of this method is based on the assumption that chemical properties of extracted solutions remain stable from the moment of collection until analysis. Extracted substrate solution can be analyzed directly in the greenhouse or sent to laboratories for complete nutritional analysis; thus, proper sample preservation methods (e.g., filtration and low temperatures) are critical for reducing sample contamination or degradation during storage. However, evidence of how these preservation methods affect chemical characteristics of PT samples is limited. The objective of this study was to evaluate the effect of storage time, storage temperature, and filtration of PT samples on pH, electrical conductivity (EC), and nutrient concentrations from pine bark– and peat-based substrates. PT extracts were obtained from liquid-fertilized fallow pots of either 100% milled pine bark (Expt. 1) or a 4 sphagnum peat: 1 perlite (by volume) substrate (Expt. 2). Aliquots of PT extract were either filtered or nonfiltered and then stored in plastic bottles at −22, 4, or 20 °C. EC, pH, and nutrient concentrations were analyzed at 0, 1, 7, and 30 days after PT sample collection. EC and pH in PT extracts of peat and pine bark, respectively, changed 1 day after collection. Storage time had the greatest effect on nutrient concentrations of samples stored at 20 °C. However, at day 30, nutrient concentrations had also changed in samples stored at 4 and −22 °C. Analytes that fluctuated most in both experiments and across all preservation treatments were dissolved organic carbon, total dissolved nitrogen, NO3−-N, and PO43−-P, whereas Ca2+, Mg2+, and SO42−-S were more stable in PT samples. This research suggests EC and pH should be analyzed immediately, whereas samples requiring nutrient analysis should be filtered immediately after collection, stored at 4 or −22 °C (preferably −22 °C), and analyzed within 7 days of collection.

Determination of gastrodin activity inhibition on acetylcholinesterase by capillary electrophoresis

Alzheimer's disease (AD) is the most common cause of dementia in elderly individuals. Currently, acetylcholinesterase inhibitors (AChEI) are the most effective clinical treatment for AD. AChEIs in natural products may have therapeutic potential and should be screened for use in AD treatment. The authors describe a simple and reliable method for AChEI screening by capillary electrophoresis (CE). A hexadimethrine bromide (HDB) solution was pushed into a capillary (0.015 MPa×10 s) and incubated for 5 min. The capillary was flushed with deionized water for 5 min to remove free HDB, followed by plugging with an acetylcholinesterase (AChE) solution. After a 5 min incubation, the AChE was immobilized on the positively charged coating by ion binding, and the micro-reactor was created. The substrate solution, acetylthiocholine iodide (AThC), was injected into the capillary and incubated in the micro-reactor for 1 min. The unreacted substrate and the enzymolysis product were separated by CE. Gastrodin, an important component of Gastrodia elata, can inhibit AChE activity. After a certain amount of gastrodin was spiked into the substance solution, the peak area of the product decreased. Greater peak area reduction indicated stronger inhibition of AChEI. We observed good reproducibility of the product peak, with relative standard deviation (RSD) values less than 5.3%. The micro-reactor can be reused up to 300 times, which greatly improves efficiency. When the concentration of gastrodin was 5.24 μmol/L, the inhibition rate of AChE reached 64.8%. The IC50 of gastrodin was (2.26±0.14) μmol/L (R2=0.9983), which was consistent with the result of traditional UV method (2.09±0.18 μmol/L). If the function of the micro-reactor deteriorates, it can be conveniently renewed by flushing the column to remove the enzyme and repeating the AChE immobilization protocol. The proposed method is simple, efficient, and low cost, and can be used to screen AChEI from natural products, thus contributing to the improvement of AD treatment.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h–1 L–1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

The potentiality of bioethanol production from corn (Zea mays L.) as a renewable source

Corn (Zea mays L.) is one of the versatile crop which is used as food, feed, fodder and in recent past as a source of bio-fuel. The sub-tropical climate is very favorable for corn cultivation. Traditionally, corn was grown in South and Southeast Asia primarily as a subsistence food crop. Worldwide it is being cultivated in over 170 countries representing an area of 185 million ha with a productivity of 5.62 t ha-1 (FAO, 2017). Out of world corn production of 1037 million MT, SAARC countries comprising of Afghanistan, Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka represent 3.2 % with a productivity of 3.8 t ha-1. Among SAARC countries, the highest productivity of 6.9 t ha-1 is reported in Bangladesh. Corn can be an important renewable source for bioethanol production. This research was carried out to evaluate Bangladeshi Corn for optimum bioethanol production. A 100 g of corn flour was mixed with 300 ml distilled water was blended and sterilized. The experiment was conducted with a temperature of 30 oC, pH 6.0 and 20 % sugar concentration. For alcoholic fermentation, 200 ml yeast (Saccharomyces cerevisiae CCD) was added to make the total volume 500 ml. Addition of small amount of 1750 unit α-amylase enzyme to the substrate solution was found to enhance the fermentation process quicker. After 6-days of incubation time corn produces 63.57 ml of ethanol with 13.33 % (v/v) purity. The non-filtered solution produces comparatively more ethanol (63.57 ml with 13.33 % purity) than the filtered solution (53.66 ml with 10 % purity). The purity can be increased by re-distillation process.

Bioethanol Production from Corn, Pumpkin and Carrot of Bangladesh as Renewable Source using Yeast Saccharomyces cerevisiae

Abstract Bangladesh produces a large amount of corn, pumpkin and carrots every year. To meet its huge energy demand and to lessen dependence on traditional fossil fuel these products are cost effective, renewable and abundant source for bioethanol production. The research was aimed to evaluate Bangladeshi corn, rotten carrot and pumpkin for bioethanol production. About 100 g of substrates was mixed with 300 ml distilled water and blended and sterilized. All the experiment was conducted with a temperature of 35oC, pH 6.0 and 20% sugar concentration. For fermentation, 200 ml yeast (Saccharomyces cerevisiae CCD) was added to make the total volume 500 ml. Addition of small amount of 1750 unit α-amylase enzyme to the substrate solution was found to enhance the fermentation process quicker. After 6- days of incubation, corn produced 63.00 ml of ethanol with 13.33 % (v/v) purity. Bioethanol production capacity of two different local varieties of pumpkin (red and black color) was assessed. Red pumpkin (Cucurbita maxima L.) produces 53 ml of ethanol with purity 6 %v/v and black color pumpkin produces 40 ml of yield with a low purity 4 %v/v. Carrot (Daucus carota L.) produces 73.67 ml of ethanol with 12.66 % (v/v) purity.

Fast photodegradation of rhodamine B and caffeine using ZnO-hydroxyapatite composites under UV-light illumination

Zinc oxide-hydroxyapatite composites were prepared using wet impregnation method. Firstly, a natural phosphate ore rich in silica and calcium phosphate was sieved to separate silica phase from phosphate phase. Then, through a chemical precipitation method, a pure hydroxyapatite (HAP) was obtained, which was used as a support for ZnO immobilization and applied for the photodegradation of two toxic contaminants: a transparent molecule (caffeine) and dye molecule (rhodamine B). During the present work two weight ratio percentages of zinc oxide were used: 25 wt.% and 50 wt.% of ZnO relative to HAP. The samples were characterized by X-ray diffraction (XRD), Fourier Transform Infrared (FTIR), X-ray Fluorescence (XRF), BET surface area (SBET), Scanning Electron Microscopy (SEM-EDS) and by Transmission Electron Microscopy (TEM-STEM). The immobilization of ZnO on HAP surface followed by thermal treatment at 400 °C for 2 h to get a homogenous dispersion of ZnO on the hydroxyapatite support. At high ZnO impregnation percentage, photodegradation performances of ZnO-HAP under UV illumination were fast and superior than the ZnO photocatalyst alone. The results showed that due to the presence of HAP, the conversion of both molecules became faster and greater, since it promotes the synergic phenomena of adsorption and photocatalysis. The toxicity of the treated substrate solutions obtained in the corn kernels germination test indicated a low toxicity after the photodegradation processes, probably due to a high mineralization degree.

A facile iron catalyzed cross-coupling reaction under micro-flow conditions

The iron-catalyzed cross-coupling reaction between an aromatic Grignard reagent and a haloalkane was investigated under micro-flow conditions. The reaction proceeded smoothly by the separate feeding of the substrate solutions to give the desired cross-coupling product. Compared to the previously reported batch reaction, the effect of additives was more predominant in the micro-flow reaction. The flow reactor system appears to contribute to the stabilization and rapid trapping of the unstable catalytic species.

Highly atom-economical and environmentally friendly synthesis of LiMn0.8Fe0.2PO4/rGO/C cathode material for lithium-ion batteries

Due to its flexible two-dimensional layered structure and good hydrophilicity, graphene oxide (GO) is widely used to prepare composite electrode materials for lithium-ion batteries by co-precipitation in aqueous solutions. However, the purification process of GO prepared by the Hummers method is cumbersome and consumes a large amount of water. It also produces wastewater containing heavy metal manganese ions. What's more, both GO and manganese compounds are used during the synthesis of LiMn0.8Fe0.2PO4/reduced graphene oxide/carbon (LiMn0.8Fe0.2PO4/rGO/C) composite. In view of the issues above, a highly atom-economical route based on Hummers method is proposed in this paper to synthesize LiMn0.8Fe0.2PO4/rGO/C. The GO solution obtained by Hummers method is directly used as a substrate solution to prepare (Mn0.8Fe0.2)3(PO4)2·xH2O/GO precipitate, which is then used as a precursor to prepare LiMn0.8Fe0.2PO4/rGO/C by high temperature solid phase sintering. The obtained particles are evenly distributed and wrapped by a graphene-based three-dimensional conductive network. The synthesized composite exhibits good rate performance and cycling stability. In a word, this research develops a new route with high atomic economy and environmental friendliness for the large-scale production of LiMnxFe1-xPO4/rGO/C composites.

La biofertilización y nutrición en el desarrollo de plántulas de chile serrano

La producción de plántulas de calidad es primordial para que durante el trasplante se propicie un crecimiento óptimo y buenos rendimientos del cultivo; por ello, el objetivo de la presente investigación fue determinar el efecto de bacterias promotoras de crecimiento vegetal y el manejo de la nutrición en la fisiología y crecimiento de plántulas de chile serrano. En condiciones de invernadero se llevó a cabo un experimento, con chile serrano var. Tampiqueña 74, bajo un diseño completamente al azar con arreglo factorial de 2×2×2. Los factores fueron: inoculación (con o sin Pseudomonas tolaasii), sustrato (con y sin esterilizar), y solución nutritiva (Steiner y té de vermicompost) con un total de ocho tratamientos con cuatro repeticiones cada uno. Los resultados indican que el efecto de P. tolaasii es altamente significativo en sustrato no esterilizado y con solución Steiner, donde las plántulas presentaron mayor altura, diámetro del tallo, área foliar, y la biomasa seca de parte aérea. Además, aumentó la concentración de NO3 en savia. Con el té de vermicompost la inoculación solo impactó en la longitud y volumen radical; mientras que con la solución Steiner únicamente tuvo efecto en el contenido nutritivo de N y K+ e índices de crecimiento. La inoculación de P. tolaasii en plántulas de chile serrano favorece la fisiología y concentración nutritiva siempre y cuando no se esterilice el sustrato. El menor efecto sobre las plántulas ocurre si sólo se suministra té de vermicompost.

Graphite-Embedded High-Performance Insulated Metal Substrate for Wide-Bandgap Power Modules

Emerging wide-bandgap (WBG) semiconductor devices such as silicon carbide (SiC) metal-oxide semiconductor field-effect transistors (MOSFETs) and gallium nitride high-electron-mobility transistors can handle high power in reduced semiconductor areas better than conventional Si-based devices owing to superior material properties. With increased power loss density in a WBG-based converter and reduced die size in power modules, thermal management of power devices must be optimized for high performance. This article presents a graphite-embedded insulated metal substrate (thermally-annealed-pyrolytic-graphite-embedded insulated metal substrate-IMSwTPG) designed for WBG power modules. Theoretical thermal performance analysis of graphite-embedded metal cores is presented, with design details for IMSwTPG with embedded graphite to replace a direct-bonded copper (DBC) substrate. The proposed IMSwTPG is compared with an aluminum nitride-based DBC substrate using finite-element thermal analysis for steady-state and transient thermal performance. The solutions' thermal performances are compared under different coolant temperature and thermal loading conditions, and the proposed substrate's electrical performance is validated with static and dynamic characterization. Using graphite-embedded substrates, junction-to-case thermal resistance of SiC MOSFETs can be reduced up to 17%, and device current density can be increased by 10%, regardless of the thermal management strategy used to cool the substrate. Reduced transient thermal impedance of up to 40% of dies owing to increased heat capacity is validated in transient thermal simulations and experiments. The half-bridge power module's electrical performance is evaluated for on-state resistance, switching performance, and switching loss at three junction temperature conditions. The proposed substrate solution has minimal impact on conduction and switching performance of SiC MOSFETs.

Dual-Purpose Inoculants and Their Effects on Corn Silage.

This study was conducted to screen dual-purpose lactic acid bacteria (LAB) from uncontrolled farm-scale silage, and then we confirmed their effects on corn silage. The LAB were isolated from eight farm-scale corn silages, and then we screened the antifungal activity against Fusarium graminearum and the carboxylesterase activity using spectrophotometer with p-nitrophenyl octanoate as substrate and McIlvane solution as buffer. From a total of 25 isolates, 5M2 and 6M1 isolates were selected as silage inoculants because presented both activities of antifungal and carboxylesterase. According 16S rRNA gene sequencing method, 5M2 isolate had 100.0% similarity with Lactobacillus brevis, and 6M1 isolate had 99.7% similarity with L. buchneri. Corn forage was ensiled in bale silo (500 kg) for 72 d without inoculant (CON) or with mixture of selected isolates at 1:1 ratio (INO). The INO silage had higher nutrient digestibility in the rumen than CON silage. Acetate was higher and yeasts were lower in INO silage than in CON silage on the day of silo opening. In all days of aerobic exposure, yeasts were lower in INO silage than CON silage. The present study concluded that Lactobacillus brevis 5M2 and L. buchneri 6M1 confirmed antifungal and carboxylesterase activities on farm-scale corn silage.

SUN-LB95 Developing a Highly Equivalent Non-Competitive Chemiluminescence Immunoassay Aldosterone Measurement to LC/MS

Background Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents anddetecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results even if measured by inaccurateradioimmunoassay, or we must accept high-cost LC/MS, and development of a more rapid and accurate substitute has been long hoped. We havedeveloped a novel, fully-automated, high-quantitative noncompetitive chemiluminescence immunoassay (NC-CLEIA) for detecting aldosterone inserum and plasma, and its performance is evaluated as compared to LC/MS measurement. Methods Recently a unique anti-metatype antibody,which recognizes the immunocomplex of aldosterone and its monoclonal antibody, was established. Using this antibody for sensing permittedthe construction of non-competitive assay for the detection of aldosterone. The reaction protocol of novel aldosterone assay is the following. Inthe 1st reaction, aldosterone in patient’s sample is captured on anti-body coated magnetic particles. Alkaline phosphatase-conjugated antimetatypeantibody is added and incubated as 2nd reaction following a wash. Then substrate solution is added after washing immunocomplex.The resulting reaction signals are proportional to the amount of aldosterone in the sample allowing quantitative determination of in serum orplasma sample. The overall reaction is completed within 30 min. Results Limit of blank (LoB), limit of detection (LoD) and limit of quantitation(LoQ) of our NC-CLEIA aldosterone assay were 0.09 ng/dL, 0.21 ng/dL and 0.57 ng/dL, respectively. NC-CLEIA aldosterone measurements werelinearly well correlated with LC/MS aldosterone measurements (N = 130, y = 1.027x - 0.23 ng/dL, Spearman’s ρ = 0.996, P< 0.0001). Bland-Altmanplot analysis between NC-CLEIA and LC-MS/MS of aldosterone revealed a bias of 0.40 ng/dL with the limits of agreement of -4.60 and 5.41 ng/dLwith 95% confidence interval. Conclusion Our novel NC-CLEIA aldosterone assay was well-correlated and had only a very low bias with LC-MS/MSmethod and also was able to accurately quantify low level samples even in essential hypertension patients. This aldosterone assay can be a most equivalent to LC-MS/MS measurement with a low cost of 12 $ and a short measuring time of 30 minutes.

Intercalation of Porphyrin‐Based SURMOF in Layered Eu(III) Hydroxide: An Approach Toward Symbimetic Hybrid Materials

AbstractA strategy for rational design of synergetic hybrid materials exploiting stabilization of intercalated layered matrices via coordination bonding is described. A new hybrid material is assembled through subsequent intercalation of the surface‐anchored metal–organic framework (SURMOF) components, zinc acetate and 5,10,15,20‐tetrakis(4‐carboxyphenyl)‐porphyrin‐zinc(II) (ZnTCPP), into the layered europium(III) hydroxychloride (LEuH). The formation of the SURMOF clusters intercalated in LEuH is confirmed by X‐ray diffraction, FTIR and Raman spectroscopy, and BET nitrogen absorption methods. The catalytic function of the SURMOF/LEuH hybrid and its components in the model reaction of hydrolysis of bis(4‐nitrophenyl) phosphate in the acidic solution is studied by UV–vis and MALDI‐TOF spectroscopy. Both the non‐intercalated matrix and the MOF powder are inactive and unstable in the substrate solution. Unlike its components, the SURMOF/LEuH hybrid exhibits synergetic catalytic activity increasing with the amount of the intercalated compounds because of the mutual stabilization of the components through coordination interactions. The results provide a basis for symbimetic (mimicking the symbiotic behavior in biological systems) hybrid materials, in which stabilization of functional units in the intercalated structure translates into a synergy of useful properties.

Multi-Channel Immunoassay System with Electrochemical Detection Toward Point-of-Care Diagnostics Using Magnetic Nanoparticles

Multi-Channel Immunoassay System with Electrochemical Detection Toward Point-of-Care Diagnostics Using Magnetic Nanoparticles

Effect of intentional convection on the passivity of an Fe–6Cr surface in pH 4.5 Na2SO4 solution

The anodic oxidation behavior of Fe–6Cr during potentio-dynamic polarization in pH 4.5 Na2SO4 solution was investigated by electrochemical ellipso-microscopy. Active, passive, and trans-passive states of the Fe–6Cr surface showed heterogeneous oxidation behaviors depending on the substrate micro-structure, electrode potential and solution convection. Enhancement of mass-transport by solution injection in the latter half of the active state resulted in acceleration of passivation of the Fe–6Cr surface. The passivity-current density of the surface passivated by intentional convection was reduced to almost half of the current density of the surface that had been in the passive state in a stagnant solution, and the surface passivated by intentional convection showed better resistance against pitting in a chloride-containing solution. The improvement of passivity of the surface was attributed to the formation of a Cr-enriched passive film by a decrease in the thickness of the diffusion layer in the latter half of the active state.

Growth characteristics of BaxSr(1−x)TiO3 thin films produced by micro-arc oxidation

Abstract

The Preparation and Characterization of Alginate–Chitosan Membranes as Solid Support for BTB and Urease Entrapment

A study of alginate-chitosan membrane synthesize was done. The membrane was prepared by mixing alginate hydrosol and chitosan hydrosol at mass ratio of 1:1 and pH of 5.28 approximately. Then it was applied for matrix immobilization of urease and bromothymol blue (BTB) by entrapment technique. The physical, chemical, thermal properties of alginate–chitosan membrane and their impact on immobilized urease activity were investigated. The polymer products were characterized by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), and Scanning Electron Microscopy (SEM). It’s showed that alginate-chitosan membrane was formed by electrostatic interaction. The obtained membran has better mechanical properties than original alginat and chitosan membranes. The immobilization urease into alginate-chitosan membrane retained the catalytic activity of the enzyme, as confirmed by color change of BTB indicator after membrane was immersed in substrate solution (urea). Therefore, alginate-chitosan membrane has good characteristics as matrix of enzyme immobilization.

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.

Vacuum lyophilization preservation and rejuvenation performance of anammox bacteria

The storage of anaerobic ammonia oxidizing bacteria (anammox) plays an important role in the application of anammox. Glycerol, sodium alginate and DMSO were used as the cryoprotectant, and vacuum lyophilization was used to prepare the anammox bacteria powder. Simultaneously, the control experiment was set up with the same protectant and preservation time. Bacteria powders were preserved using vacuum lyophilization and preserved at 4°C for 60 days. During the 54 days of rejuvenation, the reactors that were inoculated with bacteria powder preserved by different methods showed significant difference. The results show that the anammox bacteria powder with 3wt% DMSO as the cryoprotectant and without the substrate solution presented the best rejuvenation effect. The average specific anammox activity was 115.84mg-N·(g VSS·d)-1 with an activity recovery rate of 89%, and its stoichiometric ratio (Rs and Rp) was 1.33 and 0.21, which were very close to the theoretical values. The vacuum lyophilization method for the long-term preservation of anammox bacteria was effective.