Introduction 1991 1991 1997 1980 1992 8 18 1980 1992 1995 1980 1999 The aim of this study was to investigate how alkylamines are degraded by micro-organisms using a pure culture capable of utilizing alkylamines as sole carbon and energy source. Substrate specificities of the pure culture and cell-free extracts catalysing the initial degradation steps are reported. Materials and methods Chemicals ® Activated sludge Activated sludge used as inoculum was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater. Media 2 4 2 4 4 4 2 1957 −1 −1 Enrichment, isolation and growth −1 −1 −1 Culture conditions and preparation of washed cell suspensions and cell-free extracts −1 −1 Experiments on the nitrogen balance were also conducted in the fermentor fed with a mineral salts medium without ammonium chloride. The nitrogen recovery was estimated from the amount of nitrogen leaving the fermentor as biomass, ammonium and total nitrogen. The total nitrogen represents the residual substrate and/or water-soluble nitrogen-containing compound formed during the biodegradation process. g g Oxygen consumption −1 Enzyme assays All enzyme assays were performed at 30°C. Spectrophotometric enzyme assays were carried out in a Shimadzu UV 160A spectrophotometer (Shimadzu, Kyoto, Japan) in 1 cm light-path cuvettes. The oxygen uptake was measured with a polarographic oxygen monitor (Yellow Springs Instruments). −1 −1 −1 ® −1 Aldehyde dehydrogenase (NAD(P)-dependent) activity was determined by measuring the increase in absorbance at 340 nm. Reaction mixtures contained phosphate buffer (15 mM; pH 7), 0.15 μM aldehyde, 1.0 mM NAD and 0.1 mM KCN and enzyme solution in a volume of 3 ml. Analyses 1978 Protein was quantified by bicinchoninic acid method. Cells were first lysed by incubating at 95°C with 1.0 M NaOH. The protein concentration was estimated by using the Bio-Rad Protein assay kit with bovine serum albumin as standard protein. Dissolved organic carbon and total nitrogen were quantified with a Shimadzu TOC apparatus (Shimadzu). −1 g Results Isolation and characterization m d d l Pseudomonas Pseudomonas putida P. plecoglosscida P. alcaligenes Pseudomonas Growth Pseudomonas Pseudomonas Pseudomonas Depletion of dodecylamine to non-detectable concentrations from the nitrogen-free mineral salts medium in batch cultures and the concurrent growth of the isolate clearly demonstrate that this isolate also used alkylamines as its nitrogen source (data not shown). −1 Pseudomonas Respiration experiments 1 Pseudomonas Pseudomonas Pseudomonas 1 Table 1 Oxidation of various potential intermediates of alkylamine degradation by washed cell suspensions of strain BERT grown on octylamine, octanal, octanoate and acetate Substrate Growth substrate Octylamine Octanal Octanoate Acetate −1 −1 Octylamine 85 25 44 0 Octanal 62 43 66 0 Octanoate 84 53 90 41 Acetate 39 35 41 50 2 −1 −1 2 −1 −1 2 2 Table 2 Oxidation of various alkylamines by washed cell suspensions and alkylamine dehydrogenase (PMS-dependent) Substrate Washed cell suspension Cell-free extract −1 −1 Butylamine 14 5 Hexylamine 88 30 Octylamine 85 33 Decylamine 76 34 Dodecylamine 67 31 Tetradecylamine 57 35 Hexadecylamine 31 18 Octadecylamine 12 5 Nonylamine 85 35 Propylamine 7 3 Methylamine 0 0 Didecylamine 0 0 Decyldimethyamine 0 0 Decyltrimethylammonium  0 0 Dodecyl-1,3-diaminopropane 16 5 2 −1 −1 2 −1 −1 Enzymatic activities 1 −1 −1 2 2 −1 −1 Fig. 1 open square filled square −1  Discussion 1980 1992 1995 Pseudomonas 1980 Pseudomonas Pseudomonas Pseudomonas Rhodococcus 1999 alkyl alkyl Acinetobacter calcoaceticus 1992 Pseudomonas 1994 2 1 1996 1980 1995 Fig. 2 Pseudomonas  1980 1990 1988 1992 1990 2 2000 Pseudomonas 1988 P. putida 1998 Pseudomonas Pseudomonas 12 18 −1 Pseudomonas 1992 1999 Pseudomonas 1980 1995 1980