Utilization of Organic Nitrogen Compounds by Marine and Freshwater Bacteria

Reduced nitrogen species represent the largest pool of nitrogen in many aquatic systems, yet little is known about the chemical composition of this pool and the metabolic pathways required for its remineralization. The long residence time of this material in aquatic systems suggests that it is very refractory and relatively resistant to degradation by direct metabolism or extra-cellular enzymatic attack. The two objectives of this project were (1) to identify organisms with the capability to degrade representative DON compounds, and (2) to examine the ability of ammonia-oxidizing bacteria to remineralize the nitrogen from the same DON substrates. The DON substrates included: urea, trimethylamine (TMA), N-acetyl glucosamine (NAG – for ammonia-oxidizer study only), uric acid, alanine and caffeine. Enrichments on all DON substrates were successful in sustaining microbial growth and colonies were observed and isolated on agar plates of most substrates. Liquid isolations were more successful but cannot be considered “pure” at this stage. Growth experiments were conducted with pure cultures of Nitrosospira briensis and Nitrosococcus oceanii on all DON substrates. Both ammonia oxidizers were able to grow on all DON substrates (except alanine). Surprisingly fast growth rates were observed for Nitrosospira growing on urea or uric acid. In conclusion, the ability of marine and freshwater bacteria to metabolize reduced organic nitrogen substrates has been vastly underestimated and merits further study. Introduction Reduced nitrogen species represent the largest pool of nitrogen in many aquatic systems, yet little is known about the chemical composition of this pool and the metabolic pathways required for its remineralization. The concentration of this pool is determined by analysis of total dissolved nitrogen and subsequent subtraction of the inorganic components (ammonia (NH4) nitrite (NO2) and nitrate (NO3)). No investigations to date have been able to separate DON from the larger pool of organic matter for composition or structural analysis. The source of DON is presumed to bio-molecules such as peptidoglycan, a primary component of cell walls; proteins; amino-sugars and waste products such as urea. The long residence time of this material in aquatic systems suggests that it is very refractory and relatively resistant to degradation by direct metabolism or extra-cellular enzymatic attack. Nonetheless, this material must be degraded (albeit slowly) by microbes since the DON concentrations are not constantly increasing in the world’s oceans and lakes. The primary organisms responsible for large-scale nitrogen oxidation are the ammonia-oxidizing bacteria, which use the energy from the conversion of ammonia (NH3/NH4) to nitrite (NO2) to drive cellular function and biosynthesis. The amount of nitrogen required for energy production is significantly larger than that needed for protein or nucleic acid biosynthesis and so nitrogen-oxidizing bacteria could represent a large sink for reduced nitrogen. However, no bacteria have been isolated that oxidize reduced organic nitrogen for energy production (to my knowledge), although some ammonia-oxidizing bacteria have been shown to use urea as an energy source by conversion of urea to ammonia using urease. Few additional substrates were tested and so an examination of the ability of ammonia-oxidizing bacteria to remineralize a range of possible DON substrates was needed. The substrates used in this study could be divided into three groups of chemical nitrogen. First, urea and N-acetyl glucosamine (NAG) were chosen to represent amide-N. Urea is a common waste product and NAG is a 2005 Microbial Diversity course final report 8/1/05 Page 2/7 building block of peptidoglycan, the primary constituent of bacterial cell walls. Second, trimethylamine (TMA) and alanine were chosen to represent amine-N. TMA is a common waste product and alanine is a common amino acid. Lastly, caffeine and uric acid were chosen to represent aromatic-N. Both caffeine and uric acid are purine derivatives with four nitrogen atoms within two fused aromatic rings. Caffeine is commonly found in urban wastewater due to the high loading in urban sewage. Uric acid is a common component of bird guano and is routinely deposited on aquatic surfaces. In addition to testing the ability of ammonia-oxidizing bacteria ot degrade these compounds, I enriched seawater and freshwater samples with five of these compounds (i.e., all except NAG) to isolate novel bacteria that could remineralize DON. In general, these enrichments will select organisms that (1) use DON as a carbon source, (2) use DON as a nitrogen source for biosynthesis, (3) use DON as a nitrogen source for oxidation, or (4) use DON as both a carbon and nitrogen source. All six of these compounds are found in the two aquatic systems that were chosen for bacterial enrichment and thus should be representative of some of the compounds encountered by marine or lacustrine bacteria on Cape Cod. Thus, this project represents an infinitesimally small step forward in the quest to identify organisms that can degrade DON and to identify the requisite physiology for these transformations. In brief, the two objectives of this project were: 1. to identify organisms with the capability to degrade representative DON compounds, and 2. to examine the ability of ammonia-oxidizing bacteria to remineralize the nitrogen from the same DON substrates. Project #1: Water samples were acquired from John’s Pond (a freshwater pond Mashpee MA) and Garbage Beach (Vineyard Sound seawater Woods Hole MA). I set up enrichments for each environment with one DON substrate provided as both the carbon and nitrogen sources. Each seawater enrichment contained 75% autoclaved 0.2-μm filtered seawater, 0.1% trace metal mix, and 15uM PO4(all in sterile Milli-Q water). Each freshwater enrichment contained freshwater base (NaCl, MgCl2, CaCl2, KH2PO4, and KCl), 25uM SO4 10mM MOPS buffer (pH 7.2), 15uM PO4 and 0.1% trace metal mix. The concentration of DON substrate was 5 mM in each enrichment. Each enrichment was started on the same day as the sample was collected. Each sample was diluted by 10X into eight tubes of 4.5mL of enrichment medium. All tubes were placed in racks and shaken at 27C. After 9 days, agar plates were prepared for each enrichment by adding 1.5% agar to previously prepared media and then autoclaving. The enrichment dilution with the best evidence for growth was chosen for further purification (usually the 10 dilution for the freshwater enrichments and either the 10 or 10 dilutions for the seawater enrichments). Two 100uL aliquots of each dilution were transferred to appropriate agar plates for spreading and the plates were incubated at 30C in the dark. At the same time, three 10X dilutions were made of the same original enrichments into fresh media and incubated at room temperature in the dark. After three days, colonies were observed on the seawater caffeine (SWC), alanine (SWA), TMA (SWT) and urea (SWU) plates. Likewise colonies were observed on the freshwater caffeine (FWC) and alanine (FWA) plates. For both the FWC and FWA plates, the colonies were quite small, suggesting that the bacteria may have been growing on the agar instead of on the DON substrate that was added. Colonies were streak-purified from each of these plates and incubated 2005 Microbial Diversity course final report 8/1/05 Page 3/7 again. At the same time, substantial growth was observed in the liquid cultures of all the DON treatments, with the most turbid cultures observed in the alanine treatments (FWA and SWA). The presence of ammonia-oxidizing bacteria was tested in the liquid cultures. Cells were isolated from the cultures by centrifugation of 1mL aliquots in 1.8mL Eppendorf tubes (13,000 rpm for 20 min at 20C). The supernatant was removed and additional aliquots were added until a pellet was visible in the base of the Eppendorf tube. All cells were then washed in PBS:EtOH (1:1) and centrifuged again. The clean cells were resuspended in 20uL of lysis buffer (RNAse-free water with P-40 detergent) and boiled at 102C for 5 min. The resulting suspension was used for direct PCR. To test for the presence of ammoniaoxidizing bacteria, primers optimized for amoA, the gene for ammonia monooxygenase, were used to amplify the extracted DNA. Positive results were obtained for two control cultures of Nitrosospira briensis growing on either NH4 or urea. Positive results were also obtained for both the alanine treatments (SWA and FWA – Figure 1). PCR of the 16S gene (using 8F and 1492R) provided product for all treatments implying that there should have been ample template DNA in the PCR reaction for amoA amplification. Thus, it is possible to conclude that ammonia-oxidizing bacteria (or at least amoA-containing organisms) are present in the alanine amendments. Amplification of the 16S gene in these enrichments did not support this result in the sense that the most dominant organism in both the SWA and FWA enrichments were not known ammonia-oxidizers, but instead were members of the Alteromonas and Pseudomonas species, respectively. Identification of the amoA-containing organisms in these treatments will require more time and we intend to pursue the further purification of these organisms. In the end, I was able to get 16S PCR products for 11 liquid enrichments or colonies and aligned the sequences with known organisms using ARB (Figure 2). Most sequences aligned quite closely to known representatives within the ARB database. Interestingly the SWC liquid isolate sequence aligned closely with a carbazole-degrading bacterium but the plate colonies were all members of the Alteromonas group. It is possible that the plate isolations selected for different species over those in the liquid cultures. Many of the organisms observed in the cultures were quite motile and may not have grown well in the relatively immobile medium of the agar plates. Nitrosospira NH4 Urea Neg. FWUA SWA FWU FWA