Arthrobacter sp. lipase immobilization for improvement in stability and enantioselectivity

Arthrobacter sp. lipase (ABL, MTCC no. 5125) is being recognized as an efficient enzyme for the resolution of drugs and their intermediates. The immobilization of ABL on various matrices for its enantioselectivity, stability, and reusability has been studied. Immobilization by covalent bonding on sepharose and silica afforded a maximum of 380 and 40 IU/g activity, respectively, whereas sol–gel entrapment provided a maximum of 150 IU/g activity in dry powder. The immobilized enzyme displayed excellent stability in the pH range of 4–10 and even at higher temperature, i.e., 50–60°C, compared to free enzyme, which is unstable under extreme conditions. The resolution of racemic auxiliaries like 1-phenyl ethanol and an intermediate of antidepressant drug fluoxetine, i.e., ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates, provided exclusively R-(+) products (∼99% ee, E=646 and 473), compared to cell free extract/whole cells which gave a product with ∼96% ee (E=106 and 150). The repeated use (ten times) of covalently immobilized and entrapped ABL resulted in no loss in activity, thus demonstrating its prospects for commercial applications.     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Isolation and partial characterization of plasmid DNA from Arthrobacter oxidans

A method for the extraction of the high molecular weight plasmid AO 1 from the gram-positive soil bacterium Arthrobacter oxidans is presented.Following digestion of this DNA with the restriction endonucleases Accl, Bam HI, Eco RI and Hind III, an average molecular mass of 157.8 kb was estimated. This value is in good agreement with the 160 kb size determined previously by electron microscopy (Brandsch et al. 1982).Using the same method, no plasmid DNA was found in strains of the genus Arthrobacter which do not degrade nicotine, e.g., A. albidus, A. globiformis and A. auricans.Abbreviations EDTA ethylenediaminetetraacetic acid - Kb kilobasepairs - SDS sodium dodecyl sulfate - Tris Tris-(hydroxymethyl)-aminomethan     

Cloning and characterization of a cyclohexanone monooxygenase gene from Arthrobacter sp. L661

Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ɛ-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4°C. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.     

Some Properties of the Xylose/Glucose Isomerase of Immobilized Arthrobacter sp. Cells

The study of the xylose/glucose isomerase–containing Arthrobacter sp. B-5 cells immobilized in cobalt hydroxide gel showed that immobilization increases the substrate affinity of the enzyme, its thermo- and pH-optima of action and stability, and makes the addition of stabilizing cobalt ions to the isomerization medium unnecessary.     

Microbiological degradation of pentane by immobilized cells of <Emphasis Type="Italic">Arthrobacter sp.</Emphasis>

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42days of incubation at 20°C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85–95% pentane degradation during a 2month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.     

一株尼古丁降解菌株Arthrobacter sp.D4的分离鉴定及其降解代谢途径

【背景】烟草在生产和加工中会产生高浓度的尼古丁废弃物,对环境造成较大的污染。【目的】筛选降解尼古丁的微生物菌种并解析其降解尼古丁的代谢途径,理解微生物如何降解尼古丁。【方法】用常规分离筛选方法、结合形态学观察和分子鉴定手段分离和鉴定菌株类别,进而利用单因素试验方法,通过设置不同的尼古丁浓度、温度和pH确定菌株降解尼古丁的最适发酵条件和降解率,利用气相色谱-质谱联用技术检测菌株在尼古丁降解过程中的主要代谢产物。【结果】获得一株以尼古丁为唯一碳源和氮源的节杆菌属(Arthrobacter)菌株,编号为D4;该菌株降解尼古丁的最适温度和pH分别为30.0℃和7.0;在1 g/L的尼古丁浓度下具备较快的尼古丁降解速率,培养18 h时尼古丁降解率可达到90%以上;尼古丁浓度≥4 g/L时菌株生长受到明显抑制;与目前报道的节杆菌属降解途径不同,该菌株降解尼古丁过程中产生了新的终产物N-甲基吡咯烷酮、可替宁及中间产物麦斯明。【结论】本研究分离鉴定到一株具有较快尼古丁降解速率的节杆菌,该菌株很可能存在新的尼古丁降解途径。     

Degradation of 2,4,6-Trinitrophenol (TNP) by Arthrobacter sp. HPC1223 Isolated from Effluent Treatment Plant

Arthrobacter sp. HPC1223 (Genebank Accession No. AY948280) isolated from activated biomass of effluent treatment plant was capable of utilizing 2,4,6 trinitrophenol (TNP) under aerobic condition at 30 °C and pH 7 as nitrogen source. It was observed that the isolated bacteria utilized TNP up to 70 % (1 mM) in R2A media with nitrite release. The culture growth media changed into orange-red color hydride-meisenheimer complex at 24 h as detected by HPLC. Oxygen uptake of Arthrobacter HPC1223 towards various nitro/amino substituted phenols such as dinitrophenol (1.2 nmol/min/mg cells), paranitrophenol (0.9 nmol/min/mg cells), 2-aminophenol (0.75 nmol/min/mg cells), p-aminophenol (0.4 nmol/min/mg cells), phenol (0.56 nmol/min/mg cells) and TNP (2.42 nmol/min/mg cell) was analysed, which showed its additional characteristic of broad substrate catabolic capacity. The present study thus report a novel indigenous bacteria isolated from activated sludge utilized TNP and has broad catabolic potential towards substituted phenols.     

Genetic analysis of phenylacetic acid catabolism in <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> CECT386

Arthrobacter oxydans CECT386 is a Gram-positive bacterium able to use either phenylacetic acid or phenylacetaldehyde as the sole carbon and energy source for aerobic growth. Genes responsible for the catabolism of these compounds have been located at two chromosomal regions and were organized in one isolated paaN gene and two putative paa operons, one consisting of the paaD, paaF, tetR and prot genes, and one consisting of the paaG, paaH, paaI, paaJ, paaK and paaB genes. The identity of the paaF and paaN genes was supported by functional complementation experiments. A comparison with the paa catabolic genes and/or gene clusters of other bacteria that degrade these aromatic compounds is presented. The results of this study broaden the knowledge regarding the range of metabolic potential of this strain and eventually make it attractive for environmental applications.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

Evidence for two functionally distinct hydrogenases in Anacystis nidulans

The effect of growth conditions on aerobic and anaerobic hydrogenase activities of Anacystis nidulans was studied. It was found that the two hydrogenase activities both of which were confined to the particulate fraction of cell-free extracts correlated in an opposite way with growth temperature: The algae were always grown photoautotrophically in presence of H₂ but after growth at 25° C a significant oxyhydrogen reaction contrasted with negligible photoreduction rates while the opposite was true after growth at 40°C. A similar correlation between incubation temperature and induction of the respective hydrogenase activity was also observed with resting cells.Kinetic analysis of the two different types of hydrogenase — catalysed reactions with Anacystis membranes yielded the following Michaelis-Mentenparameters: K _M=55 M H₂ and v _max=0.12 mol H₂ per min and mg protein for the oxyhydrogen reaction, and K _M=170 M H₂ and v _max=0.3 mol H₂ per min and mg protein for the photoreductions. Also the dependences of oxyhydrogen and of photoreduction activities on pH and on temperature were measured; both pH and temperature profiles were found to be markedly different for each type of H₂-supported reaction.The results are discussed as pointing to the possible occurrence of two functionally distinct hydrogenase enzymes which can be synthesized by Anacystis in response to the conditions of induction.Abbreviations BO p-benzoquinone - CAP chloramphenicol - chl chlorophyll - cytc horse heart cytochrome c - DCMU 3-(34-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - fd ferredoxin - FeCy ferricyanide - MB methylene blue - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethan     

Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli

An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg²⁺. The enzyme was poly -D-1, 4-mannuronate-specific rather than -D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly -D-1, 4-mannuronate-specific alginate lyase from the same strain.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

Isolation and characterization of a fructosyl-amine oxidase from an <Emphasis Type="Italic">Arthrobacter </Emphasis>sp.

An Arthrobacter sp. was isolated that, when induced by fructosyl-valine, expressed a fructosyl-amine oxidase (FAOD) that was specific for -glycated amino acids. The N-terminal amino acid sequence of the purified oxidase was determined and used to design oligonucleotides to amplify the gene by inverse PCR. Expression of the gene in Escherichia coli produced 0.23 units FAOD per mg protein, over 30-fold greater than native expression levels, with properties almost indistinguishable from the native enzyme. The presence of FAOD was confirmed in other Arthrobacter ssp.Revisions requested 8 September 2004; Revisions received 4 November 2004     

Mineralization of 4-aminobenzenesulfonate (4-ABS) by Agrobacterium sp. strain PNS-1

A bacterial strain, PNS-1, isolated from activated sludge, could utilize sulphanilic acid (4-ABS) as the sole organic carbon and energy source under aerobic conditions. Determination and comparison of 16S r DNA sequences showed that the strain PNS-1 is closely related to the species of Agrobacterium genus. Growth on 4-ABS was accompanied with ammonia and sulfate release. TOC results showed complete mineralization of sulphanilic acid. This strain was highly specific for 4-ABS as none of the sulphonated aromatics used in the present study including other ABS isomers were utilized. Strain PNS-1 could, however, utilize all the tested monocyclic aromatic compounds devoid of a sulfonate group. No intermediates could be detected either in the growth phase or with dense cell suspensions. Presence of chloramphenicol completely inhibited 4-ABS degradation by cells pregrown on succinate, indicating that degradation enzymes are inducible. No plasmid could be detected in the Agrobacterium sp. Strain PNS-1 suggesting that 4-ABS degradative genes may be chromosomal encoded.     

Morphological,physiological and enzymatic characteristics of cephalosporin acylase-producing Arthrobacter strain 45-8A

A bacterial strain producing cephalosporin acylases was isolated from soil. The morphological and physiological properties of this strain suggest that it belongs to the genus Arthrobacter, and the isolate was therefore designated Arthrobacter strain 45-8A. Substrate specificity of the enzyme was examined. The enzyme can convert both cephalosporin C and 7-(4-carboxylbutan-amino)cephalosporanic acid to 7-aminocephalosporanic acid. An interesting feature of the acylases is their temperature-dependent regulation. Activity of acylases was detected in strain 45-8A grown at temperature below 30 °C, but was not observed at higher temperature. Arthrobacter strain 45-8A did not exhibit -lactamase activity, even though its resistance to cephalosporin C was very strong (>2000 g/ml). This is quite beneficial for its application in the manufacture of 7-aminocephalosporanic acd.Abbreviations used NBHAB 2-Nitro-5-(6-bromohexanoylamino)-benzoic acid - NIPAB 2-Nitro-5-phenylacetaminobenzoic acid - CPC cephalosporin C - GL-7ACA 7-(4-carboxybutanamino)cephalosporanic acid - 6-APA aminopenicillanic acid - 7-ACA 7-aminocephalosporanic acid - PDAB p-Dimethylaminobenzaldehyde     

Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 2AC and <Emphasis Type="Italic">Comamonas</Emphasis> sp. 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75–90% of the initial TOC (total organic carbon) was mineralized, 5–20% remained as DOC (dissolved organic carbon) and 3–10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0–19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with eitherArthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.