Urea production by the marine bacteria Delaya venusta and Pseudomonas stutzeri grown in a minimal medium

The present experiment showed that the marine bacteria Delaya venusta and Pseudomonas stutzen produced urea when grown in a minimal medium without an external supply of organic nitrogen. The urea production rate depended on the bacterial state of growth, and the highest urea accumulation rates in the medium were found in the growth deceleration phase and in the beginning of the stationary phase. Urea did not accumulate in D. venusta cells, whereas the intracellular accumulation of urea in P. stutzeri cells exceeded urea accumulation in the medium during exponential growth. Further, D. venusta could, in contrast to P. stutzen, hydrolyse urea. We suggest that intracellular purines and pyrimidines (in particular RNA) were potential sources for the observed urea production. K E Y WORDS: Marine bacteria. Urea . Production . Turnover. Growth phases Urea is an important organic nitrogen compound in the marine environment (e.g. Remsen 1971, McCarthy et al. 1977, Sorensson & Sahlsten 1987, Price & Harrison 1988, Lomstein et al. 1989, Cochlan & Harrison 1991, Therkildsen & Lomstein 1994). Urea production has been related to the presence and input of readily degradable organic material (Satoh 1980, Lomstein et al. 1989, Therkildsen & Lomstein 1994) and the highest concentrations of urea have been found in the sediment surface, which is characterized by a low C/N ratio. A low C/N ratio has been used as an indicator of high quality organic matter (Blackburn 1986, Lomstein et al. 1989, Therkildsen & Lomstein 1994). Bacteria can play a significant role in the overall production of urea in marine sediments (Pedersen et al. 1993a, Therkildsen & Lomstein 1994). Purines and pyrimidines are potential precursors for bacterial urea production (e.g. Vogels & Van der Drift 1976, Busse et al. 1984, Gott'Addressee for correspondence. E-mail: bente@pop.bio.aau.dk schalk 1986, Kaspari & Busse 1986). A substrate addition experiment with anoxic, defaunated marine sediment showed a considerable urea production, when the sediment was supplemented with adenosine 5'monophosphate (AMP), cytidine 5'-monophosphate (CMP), and 16s ribosomal RNA (Therkildsen et al. 1996). However, bacterial urea production from purines and pyrimidines is even more favourable under oxic conditions (Vogels & Van der Drift 1976). Bovine serum albumin did not stimulate sediment urea production, which indicated that urea was not an important intermediate in protein degradation (Therkildsen et al. 1996). Culture experiments with Thiosphaera pantotropha showed that the bacteria produced urea during exponential growth when cultured under oxic and anoxic conditions in a minimal medium (Pedersen et al. 1993b). The present experiment was designed to investigate urea production by the marine bacteria Delaya venusta and Pseudomonas stutzeri grown without any supplements of organic nitrogen under oxic conditions. Urea production was followed during the different growth phases of the cultures. D. venusta is an aerobic bacterium belonging to the family Halomonadaceae. The uptake, transport and turnover of urea by D. venusta has been studied by Jahns (1992). P. stutzeri is a facultative, aerobic, nonfluorescent, denitrifying pseudomonad commonly encountered in diverse habitats, including marine environments, soil, and sewage (Ward & Cockcroft 1993). Methods. Delaya venusta was grown at 30°C, at pH 7.3, in a minimal medium and under oxic conditions. Pseudomonas stutzen was grown at 18"C, at pH 7.0, in a similar minimal medium and under oxic conditions. The minimal medium contained the following in g I-': Na,HPO,, 2.44; KHpP04, 1.52; (NH4)2S04, 0.5; MgSO, . 7H20, 0.2; CaC12 2H20, 0.05; NaCl, 29.22; 0 Inter-Research 1997 214 Mar Ecol Prog Ser 13: 213-217, 1997 and 5 m1 of the trace element solution described in Finster et al. (1992). Glucose was added to a final concentration of 10 mM. Samples were withdrawn from the cultures at regular time intervals to follow the change in the urea concentration in the medium, the total concentration of urea (medium+bacteria) and bacterial density. The concentration of urea in the medium was measured on 0.2 pm Nucleopore filtered samples by the diacetylmonoxime method (Mulvenna & Savidge 1992). The total concentration of urea (medium+bacteria) was measured by addition of the acid reagent from the diacetylrnonoxime method directly to unfiltered samples. The diacetylmonoxime-sample mixture was thoroughly mixed and reacted for 10 min before centrifugation at 11 000 X gfor 10 min. The remaining reagents in the Mulvenna & Savidge (1992) urea method were added to the supernatant, and the concentration of urea was measured. Bacterial density was measured as optical density on a Milton Roy, Spectronic 601 spectrophotometer at 600 nm. Additional samples were withdrawn from the cultures in the beginning and at the end of the experiment to measure the NH4+ concentration. NH,' was measured by the salicylatehypochlorite method (Bower & Holm-Hansen 1980). Urea turnover was measured twice in Delaya venusta and Pseudomonas stutzeri cultures by the 14C-urea isotope dilution technique (Lund & Blackburn 1989). Eight subsamples (2 m1 culture) were collected and transferred to 10 m1 sterile glass containers (Exetainer, Labco) in each turnover measurement. Half of the samples were used to follow the change in the concentration of urea during incubation and the remaining samples were injected with 14C-urea. The '4C-incubation was modified slightly from Lund & Blackburn (1989): (1) the incubation was performed as a 4 point time course (-0, 1.0, 1.5,3.5 h); (2) the activity of the injected 10pl of tracer was 9.6 nCi p1-' (56 mCi mmol-'; Amersham Radiochemical Center); (3) the incubation was terminated by addition of 2 m1 2.5% NaOH; (4) the scintillation fluid was pH-adjusted OPTIFLUORTM from Packard (1:lO v/v 0.1 M NaOH:OPTIFLUORTM). Urea turnover rates were calculated by the nonsteady-state model I, described in Lund & Blackburn (1989). The purity of the Delaya venusta cultures were confirmed microscopically before and after the experiment and during the experiment by the characteristic pea (Pisum sp.) odour of the cultures. Purity of the Pseudomonas stutzen' cultures were confirmed microscopically and by the rnicromethod API 20 NE for identification of nonenteric Gram-negative rods (bioMerieux) before and after the experiment. D. venusta and P. stutzeri cultures were purchased from DSM-Deutsche Sammlung von Mikroorganismen und Zeukulturen, Germany. 0.0 0 10 20 30 40 50 Time (h)