Strains and growth conditions
The A. bisporus strain A15 (Sylvan, Horst, the Netherlands) was routinely grown at 25 °C on malt extract agar (MEA; 20 g l−1 malt extract agar [BD biosciences, Franklin Lakes, USA], 2.1 g l−1 MOPS, pH 7.0, and 1.5% agar). Spawn was made by mixing pieces of colonized MEA (0.5 by 0.5 cm) with a sterilized mixture of 50 g rye, 1.4 g CaCO3, 1 g CaSO4, and 50 ml demi water. The rye was colonized in 3 weeks at 25 °C and stored at 4 °C before use. PII-end compost (CNC Grondstoffen, Milsbeek, The Netherlands) was inoculated (5 spawn grains per 25 g of compost) in 250 ml bottles and incubated in the dark at 25 °C for up to 26 days. Compost not inoculated with A. bisporus served as a control. Samples of PII-end compost and PIII compost were stored at −20 °C.
Compost agar medium (CAM) was prepared by homogenizing 75 g phase III compost (CNC Grondstoffen) in 0.5 l water. The mixture was autoclaved three times at 120 °C for 20 min and mixed 1: 1 with 3% agar. To produce pure A. bisporus mycelium CAM was overlaid with a polycarbonate (PC) membrane (diameter, 76 mm; pore size, 0.1 µm; Profiltra, Almere, the Netherlands) and inoculated at the center of the plate. After 14 days at 25 °C mycelium was lyophilized, and ground to a powder.
Compost extract was prepared by mixing 50 mg lyophilized and milled compost with 1 ml demi water and incubating head over tail for 1 h at 25 °C. Insolubles were removed by centrifugation at 4 °C and 15,000g for 15 min. Laccase activity was determined by adding 20 µl five times diluted compost extract with 1 ml 1 mM ABTS in citric phosphate buffer, pH 4. Change in absorbance was followed at 420 nm for 30 s. Activity in units (U) was calculated using the law of Lambert–Beer with an extinction factor of 36,000 M−1 cm−1.
Quantification of chitin
N-Acetylglucosamine (GlcNAc) released from chitin was quantified in technical triplicates using a colorimetric assay (Reissig et al. 1955; François 2006). Compost (20–30 mg dry weight) was mixed with 1 ml 6% KOH (w/v) and incubated at 80 °C for 90 min. The mixture was centrifuged at 10,000g at 4 °C for 10 min after adding 500 µl glacial acetic acid. Pellets were washed twice with water and once with 50 mM potassium phosphate buffer, pH 6.5. Chitin was digested overnight in 600 µl 50 mM potassium phosphate buffer pH 6.5 containing 0.42 U chitinase, 8.3 U lyticase, and 2 µl protease inhibitor (P8215, Sigma-Aldrich, St Louis, USA). Water was added to a final volume of 1.2 ml and 100 µl was used for colorimetric quantification of GlcNAc. To this end, 50 µl demi water and 150 µl 0.27 M tetraborate were added. After incubation at 100 °C for 15 min, 1.8 ml Reissig reagent (10 g 4-(dimethylamino)benzaldehyde, 12.5 ml of 10 M HCl, and 87.5 ml glacial acetic acid) was added and incubated at 40 °C for 20 min. Liquid was transferred to a cuvette and the OD585 was measured. A standard curve of 0–90 nmol GlcNAc (with a detection limit of 2 nmol) was used to calculate the release of the aminosugar per gram compost after subtracting the signal obtained from the assay in the absence of chitinase. 0.5–8 mg of pure mycelium was used to determine the amount of GlcNAc release mg−1 mycelium. The average OD of the reaction mixture in the absence of compost was substracted from the OD of mixtures containing fungal colonized compost or pure mycelium. GlcNAc was not detected when using 3 × 109 dH5α Escherichia coli cells that contain a total amount of 174 nmol of the aminosugar (Wientjes et al. 1991). This shows that the GlcNAc in the assay does not orginate from bacterial biomass.
Phospholipid fatty acid analysis
PLFAs were extracted from 300 mg compost as described (Frostegård et al. 1991, 1993; Hedlund 2002) based on the methods of Bligh and Dyer (1959) and White et al. (1979). In short, PLFAs were extracted using 10 ml Bligh and Dyer solution (CHCl3:MeOH:citrate buffer, 1:2:0.8 v/v/v). Phases were separated by addition of 4 ml CHCl3 and 4 ml citrate buffer during overnight incubation at room temperature. An aliquot of 3 ml of the lipid extract was transferred to a glass tube. After evaporation of the solvent with a stream of N2, samples were solubilized in CHCl3 and applied on a silica column. Phospholipids were eluted with 1.5 ml MeOH after eluting the neutral and glycolipids with 1.5 ml CHCl3 and 6 ml acetone, respectively. 5 µg of methyl nonadecanoate (C19:0, Sigma-Aldrich) was added to each sample as an internal standard. Samples were transesterified at 37 °C for 15 min by addition of 1 ml toluene:methanol (1:1) and 1 ml freshly made 0.2 M KOH in methanol. After cooling to room temperature for 20 min, 2 ml hexane:CHCl3 (4:1 v/v), 0.3 ml 1 M HAc, and 2 ml H2O were added. Samples were centrifuged for 5 min at 685 g and 5 µg of methyl dodecanoate (C12:0, Sigma Aldrich) was added as an internal control to the upper phase that contained the PLFAs. After evaporation of the solvent by a stream of N2, lipids were taken up in 200 µl hexane for gas chromatography-flame ionization detector analysis. PLFAs were identified based on retention time and equivalent chain length as calculated using C12:0, C16:0, and C19:0. Abundance in nmol g−1 was calculated using spiked C19:0. The C18:2ω6 marker was used to estimate fungal biomass, while various PLFA markers (Additional file 1: Table S1) were used as markers of bacterial biomass (Frostegård and Bååth 1996; Hedlund 2002; Ruess and Chamberlain 2010).
Custom R (v3.03) scripts were used for annotation of PLFAs. Statistical analysis of chitin and PLFA biomass was done with t tests in SPSS Statistics 22 software. Changes of laccase, chitin, and PLFA over time were analysed using ANOVA with Bonferroni or Dunnet T3 post hoc correction in SPSS Statistics 22 software. In all cases p was <0.05.