Geographic origin and identity of isolate An-4
The sampling site was located in the coastal, seasonal OMZ off Goa (India), northwest of the river mouths of the Zuari and the Mandovi (15°31′80″N, 73°42′60″E). Sampling was carried out at 14 m water depth in October 2005 and anoxic conditions were recorded in the bottom waters during sampling. Four ascomycete fungi were successfully isolated by the particle-plating technique after enrichment in anoxic, nitrate-amended seawater. One of the ascomycete isolates (An-4) was axenized with antibiotics and is tested here for its capability to reduce nitrate in the absence of oxygen.
Isolate An-4 was identified as Aspergillus terreus (Order Eurotiales, Class Eurotiomycetes) using morphological and DNA sequence data. Macro- and microscopic characters were studied according to
. Partial calmodulin (Cmd) and β-tubulin (BenA) gene sequences retrieved from the isolate with previously described methods
[57, 58] were used to derive the phylogenetic position of An-4 (Additional file
1: Figure S2). The obtained sequences were deposited in the NCBI GenBank sequence database under accession numbers [KJ146014] (Cmd) and [KJ146013] (BenA). The isolate was deposited in the culture collection of the CBS-KNAW Fungal Biodiversity Centre as [CBS 136781] and at the Microbial Type Culture Collection and Gene Bank (MTCC, Chandigarh, India) as [MTCC 11865].
Cultivation for anaerobic nitrate turnover experiments
An-4 was pre-grown on agar plates prepared from YMG broth (i.e., Yeast extract [8 g L-1] + Malt extract [10 g L-1] + Glucose [10 g L-1]) supplemented with penicillin and streptomycin. Every few plate transfers, the antibiotics were omitted to avoid emergence and carry-over of resistant bacteria. Spores of the axenic isolate grown on agar plates were used to inoculate 500-mL Erlenmeyer flasks that contained 250 mL of YMG broth. For aerobic cultivation, the flasks were closed with aseptic cotton plugs. The flasks were placed on a rotary shaker (120 rpm) and incubated at 26°C. Under these conditions, the mycelia of An-4 formed spherical aggregates of 2–5 mm in diameter. The transfers from plate to flask were repeated every 3–4 weeks.
Anaerobic nitrate turnover
The capability of An-4 to reduce nitrate anaerobically was investigated in two experiments: (1) An-4 was cultivated in Erlenmeyer flasks under oxic vs. anoxic conditions in the presence of both NO3
- and NH4
+, and (2) An-4 was pre-cultivated in Erlenmeyer flasks under oxic conditions in the presence of 15NO3
- and then exposed to anoxic conditions in gas-tight incubation vials.
In Experiment 1, the fate of NO3
- and NH4
+ added to the liquid media was followed during aerobic and anaerobic cultivation of An-4. Six replicate liquid cultures were prepared as described above, but with the YMG broth adjusted to nominal concentrations of 50 μmol L-1 NO3
- and 50 μmol L-1 NH4
+ using aseptic NaNO3 and NH4Cl stock solutions, respectively. Three cultures were incubated aerobically, whereas the other three cultures were incubated anaerobically by flushing the Erlenmeyer flasks with dinitrogen for 30 min and then closing them with butyl rubber stoppers. Subsamples of the liquid media (1.5 mL) were taken after defined time intervals using aseptic techniques. Anaerobic cultures were sampled in an argon-flushed glove box to avoid intrusion of O2 into the Erlenmeyer flasks. Samples were immediately frozen at −20°C for later analysis of NO3
- and NH4
In Experiment 2, the precursors, intermediates, and end products of dissimilatory nitrate reduction by An-4 were investigated in a 15N-labeling experiment, involving an oxic-anoxic shift imposed on axenic mycelia. For the aerobic pre-cultivation, a liquid culture was prepared as described above, but with the YMG broth adjusted to 120 μmol L-1
- (98 atom% 15N; Sigma-Aldrich). For anaerobic incubation, fungal aggregates were transferred to gas-tight glass vials (5.9-mL exetainers; Labco, Wycombe, UK) filled with anoxic NaCl solution (2%) amended with nitrate as electron acceptor and glucose as electron donor. Using aseptic techniques, equally-sized subsamples of fungal aggregates were transferred from the aerobic pre-cultures into 30 replicate exetainers. The wet weight of the aggregates was determined. Then the exetainers were filled with anoxic NaCl solution adjusted to 120 μmol L-1
- and 25 μmol L-1 glucose. Care was taken not to entrap any gas bubbles when the exetainers were closed with the septum cap. The exetainers were fixed in a rack that was continuously rotated to keep the aggregates in suspension and were incubated at 26°C in the dark for 24 days.
The anaerobic incubation was terminated in batches of three exetainers after defined time intervals. Subsamples of the liquid media were withdrawn through the septum (and simultaneously replaced with helium) for analyzing the concentrations of extracellular NO3
+, and N2O, while the concentrations of 15N-N2O and 15N-N2 were determined directly in the incubation exetainers. For NO3
-, and NH4
total analysis, 1.5 mL of the liquid media was immediately frozen at −20°C. For N2O analysis, 1 mL of the liquid media was immediately transferred into an N2-purged 3-mL exetainer and fixed with 100 μL ZnCl2 (50%). For 15NH4
+ analysis, 0.5 mL of the liquid media was transferred into a 3-mL exetainer and frozen at −20°C. The liquid media remaining in the incubation exetainers were fixed with 100 μL ZnCl2 (50%) for later 15N-N2O and 15N-N2 analysis. For technical reasons, 15N-N2O could not be quantified for this specific experiment, but only for a slightly modified twin experiment the results of which are presented in the Supporting Information.
Additional exetainers with fungal aggregates were prepared and treated in the same way as the other exetainers for verifying that An-4 remained axenic throughout the anaerobic incubation. At the end of the experiment, these exetainers were opened using aseptic techniques and subsamples of both fungal aggregates (at least two) and liquid medium (100 μL) were plated on YMG agar. After incubation at 26°C for 15 days, the fungal colonies were carefully checked by microscopy for the presence of bacteria and xenic fungi. All microscopic checks were negative. Additionally, DNA was extracted from fungal aggregates and liquid medium with the UltraClean™ Soil DNA Isolation Kit (Mo Bio, Carlsbad, CA) and used as template for PCR targeting the 16S rRNA gene with the universal bacterial primers GM3F/GM4R
. All molecular checks were negative, since agarose gel electrophoresis did not reveal any specific amplification product except for in the positive control, a laboratory strain of Agrobacterium sp.
Intracellular nitrate storage
The capability of An-4 to store nitrate intracellularly was investigated during both aerobic and anaerobic cultivation (Experiment 3). Liquid cultures were prepared as described above, but with the YMG broth adjusted to 50 μmol L-1 NO3
-. After defined time intervals, YMG broth and fungal aggregates were subsampled for analysis of NO3
- freely dissolved in the broth (i.e., extracellular nitrate = ECNO3) and NO3
- contained within the fungal hyphae (i.e., intracellular nitrate = ICNO3). Subsamples for ECNO3 analysis (1.5 mL) were cleared from suspended hyphae by mild centrifugation at 1000× g for 10 min and the supernatants (S0) were stored at −20°C for later analysis. Fungal aggregates for ICNO3 analysis were collected in a 2-mL centrifugation tube and the adhering YMG broth was siphoned off using a hypodermic needle. The aggregates were washed with 1 mL nitrate-free NaCl solution (2%) and blotted dry on nitrate-free filter paper. The aggregates were then equally distributed among two 15-mL centrifugation tubes, one for ICNO3 analysis and one for protein analysis.
Aggregates intended for ICNO3 analysis were weighed and thoroughly mixed with 2.5 mL nitrate-free NaCl solution (2%) and centrifuged at 1000× g for 5 min. Half a milliliter of the supernatant (S1) was stored at −20°C for later analysis. To make the fungal hyphae burst and release the ICNO3 into the NaCl solution, the tube was alternately cooled down to −196°C in liquid nitrogen and heated up to +90°C in a water bath for 5 min each. Cell disruption was additionally promoted by a 1-min treatment with an ultrasonic probe (UW70, Bandelin, Germany). The homogenized hyphae were pelleted by centrifugation at 3000× g for 10 min and the supernatant (S2) was stored at −20°C for later analysis.
Aggregates intended for protein analysis were suspended in 4 mL 0.5 M NaOH, sonicated for 1 min, and incubated at +90°C for 15 min for hot alkaline extraction of cellular proteins. The hyphae were pelleted by centrifugation at 3000× g for 5 min and the supernatant was stored at −20°C for later protein analysis according to
. Protein extraction was repeated with the pelleted hyphae and the results of the analysis of the two supernatants were combined. A conversion factor (wet weight → protein content) was derived and used for calculating the biomass-specific ICNO3 contents as the difference between NO3
- concentrations in S1 and S2 divided by the protein contents of the hyphae.
Production of biomass and cellular energy
The production of biomass and cellular energy by An-4 was studied during aerobic and anaerobic cultivation in the presence or absence of NO3
- (Experiment 4). For this purpose, the time courses of protein and ATP contents of An-4 mycelia and of NO3
- and NH4
+ concentrations in the liquid media were followed. Twelve replicate liquid cultures were prepared as described for Experiment 1, but in six cultures NO3
- addition was omitted. Six cultures (3 cultures each with and without NO3
-) were incubated aerobically, whereas the other six cultures (3 cultures each with and without NO3
-) were incubated anaerobically. Subsamples of the liquid media (1.5 mL) and An-4 mycelia (4–6 aggregates) were taken after defined time intervals using aseptic techniques. Samples were immediately frozen at −20°C for later analysis of NO3
- and NH4
+ concentrations and protein and ATP contents. The NO3
--amended cultures received additional NO3
- (to a nominal concentration of 50 μmol L-1) after 1, 3, 7, and 9 days of incubation to avoid premature nitrate depletion.
Nitrate and NO2
- were analyzed with the VCl3 and NaI reduction assay, respectively
[61, 62]. In these methods, NO3
- and/or NO2
- are reduced to nitric oxide that is quantified with the chemiluminescence detector of an NOx analyzer (CLD 60, Eco Physics, Munich, Germany). Ammonium was analyzed with the salicylate method
. Nitrous oxide was analyzed on a gas chromatograph (GC 7890, Agilent Technologies) equipped with a CP-PoraPLOT Q column and a 63Ni electron capture detector. Isotopically labeled ammonium (15NH4
+) was analyzed with the hypobromite oxidation assay
[64, 65] followed by 15N-N2 analysis on a gas chromatography-isotopic ratio mass spectrometer (GC-IRMS; VG Optima, Manchester, UK). Prior to hypobromite addition, care was taken to remove any N2 possibly produced during the anaerobic incubation by flushing with helium for 5 min. Headspace samples for 15N-N2O and 15N-N2 analysis were taken directly from the incubation exetainers and measured on the GC-IRMS.
Biomass-specific contents of adenosine triphosphate (ATP) of An-4 were determined using a modified protocol for ATP quantification in aquatic sediments
. Briefly, 1–3 pre-weighed An-4 aggregates were sonicated in 5 mL of ice-cold extractant (48 mmol L-1 EDTA-Na2 in 1 mol L-1 H3PO4) for 1 min and then stored on ice for 30 min. The cell suspension was centrifuged at 3000× g for 10 min and 1 mL of the supernatant was diluted 1:10 with autoclaved deionized water and adjusted to pH 7.8 with NaOH. An ATP assay mix (FLAAM, Sigma-Aldrich) and a luminometer (TD 20e Luminometer, Turner Designs) were used to quantify the extracted ATP with the firefly bioluminescence reaction. The ATP assay mix was diluted 1:25 with a dilution buffer (FLAAB, Sigma-Aldrich). Calibration standards (0–100 μmol L-1) were prepared from ATP disodium salt hydrate (A2383, Sigma-Aldrich) dissolved in 1:10-diluted extractant adjusted to pH 7.8. Biomass-specific ATP contents of An-4 were calculated from the ATP concentrations of the extracts and the protein contents of the An-4 aggregates.