192 Introduction After publication of the original manuscript, the presented method has been applied in various 15 N- and 13 C-labeling studies to trace incorporation of 15 N and/or 13 C into bacterial and total microbial biomass in different coastal sediments (Veuger et al. 2006, Veuger et al. 2007, Veuger and Middelburg 2007). These applications and analysis of the D-Ala content of some algal and cyanobacterial cul- tures yielded valuable information regarding the interpretation of 15 N and 13 C incorporation into D-Ala and other hydrolysable amino acids (HAAs), which is presented in this update. Calculation of D/L-Ala ratio In the original publication, the abundance of D-Ala was expressed relative to that of L-Ala as D/L-Ala ratio (%) that was calculated as D/L × 100. However, in most papers, the abundance of D- versus L-AAs is presented as D/L (i.e., not %) or as %D (D/(D + L) × 100) (e.g., McCarthy et al. 1998, Dittmar et al. 2001, Amon et al. 2001, Kaiser and Benner 2005). To prevent unnecessary confusion and for consistency, we now also use D/L. Hydrolysis-induced racemization In the original publication, it was assumed that formation of D-Ala from L-Ala by racemization during acid hydrolysis of the samples was negligible compared to the amount of D-Ala from bacteria. However, it is now clear that hydrolysis- induced racemization yields substantial amounts of D-Ala and hence should be corrected for. Recently, Kaiser and Benner (2005) showed that liquid-phase hydrolysis of proteins and algal biomass in 6 M HCl for 20 h at 110°C (same settings as used in present method) yielded D/L-Ala ratios between 0.017 and 0.019. These values are very similar to those measured for our axenic algal cultures (0.015–0.018, Table 4). Moreover, in all 15 N- and 13 C-labeling experiments so far, excess 15 N and 13 C D/L-Ala ratios were never <0.015, even for incubations of sur- face sediment slurries in the light in which algal biomass was an order of magnitude higher than bacterial biomass (Veuger and Middelburg 2007). Altogether, these results indicate that racemization during hydrolysis of microbial biomass (using hydrolysis conditions as used in the present method) results in D/L-Ala ratios of 0.015 to 0.02 (i.e., ~1.5% to 2% of L-Ala is con- verted to D-Ala). Following Kaiser and Benner (2005), we used these values to empirically correct our D-Ala data. Measured D- Ala concentrations and excess label in D-Ala can be corrected for hydrolysis-induced racemization as follows: x bacterial D-Ala = [measured x D-Ala] – (0.017 × [measured x L-Ala]) where x is the concentration of excess 15 N or 13 C; 0.017 is used as the average of the 0.015–0.02 range for hydrolysis-induced racemization. When results are presented as D/L-Ala ratios (concentrations of excess 15 N or 13 C), the most straightforward way is to present measured (i.e., uncorrected) ratios and to indicate the racemization background of 0.015–0.02 graphi- cally (see Fig. 7). Bacterial D/L-Ala ratios In the original manuscript, we adopted the common assump- tion that aquatic bacterial communities are dominated by Gram- negative (G–) bacteria and hence that the D/L-Ala ratio of these bacterial communities is similar to that for Gram negatives that appear to have a relative uniform D/L-Ala ratio of ~0.05 (previ- ously called 5%) (Table 2). However, some of the recent 15 N- and 13 C-labeling studies in sediments yielded excess 15 N and 13 C D/L- Ala ratios up to ~0.1 (Veuger et al. 2006, Veuger et al. 2007, Veuger and Middelburg 2007). These relatively high D/L-Ala ratios appear to be due to substantial label incorporation by Gram-positive (G+) bacteria and/or cyanobacteria, since these are characterized by a thicker peptidoglycan layer (Madigan et al. 2000) and a corresponding higher D-Ala content and D/L-Ala ratio. Although Gram negatives indeed appear to dominate bac- terial communities in the water column, Gram positives have been reported to contribute up to 30% of the total bacterial com- munity in anaerobic subsurface sediment (Moriarty and Hayward 1982). This relatively high G+ abundance in deeper sediment is consistent with increasing relative abundance of phospholipid- derived fatty acids (PLFAs) characteristic for Gram positives with Update of “Analysis of 15 N incorporation into D-alanine: A new method for tracing nitrogen uptake by bacteria” (Veuger et al. 2005, Limnol. Oceanogr. Methods 3:230-240) Bart Veuger, Jack J. Middelburg, Henricus T. S. Boschker, and Marco Houtekamer Centre for Estuarine and Marine Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 140, 4400 AC Yerseke, The Netherlands Limnol. Oceanogr.: Methods 5, 2007, 192–194 © 2007, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS