All of the chemicals and reagents used were purchased from the Sigma-Aldrich Co. USA, unless mentioned otherwise. The reagents, when available, were molecular biology grade. All solutions were prepared using these reagents and sterile distilled water.
Bacterial strains and culturing methods
B. sp. SGP1 was isolated from a waste water sludge sample obtained from the Hyum Ki So Hwa Jo anaerobic digestion tank, Seoul, South Korea. This strain was co-cultured with C. tyrobutyricum ATCC 25755T in reinforced clostridial media (RCM). The media was made fresh using the individual components and supplemented with 40.0 g/L of either sucrose for the co-culture tests or B. sp. SGP1 monocultures or glucose for C. tyrobutyricum ATCC 25755T monocultures. The composition of the RCM medium was as follows: beef extract (Difco, USA) 10.0 g/L, peptone (Difco, USA) 5.0 g/L, yeast extract (Difco, USA) 3.0 g/L, tryptone (Difco, USA) 5.0 g/L, starch 1.0 g/L, sodium acetate 3.0 g/L, and NaCl 5.0 g/L. The medium was purged with argon gas and aliquoted into 60 ml serum bottles (20 ml in each), after which it was sterilized by autoclaving at 121°C for 20 minutes. For some experiments, P2 medium was used instead of RCM. The composition of the P2 medium was as follows: yeast extract (Difco, USA) 1.0 g/L, K2HPO4 0.5 g/L, KH2PO4 0.5 g/L, ammonium acetate 2.2 g/L, p-aminobenzoate 0.001 g/L, thiamin 0.001 g/L, biotin 0.00001 g/L, MgSO4.7H2O 0.2 g/L, MnSO4.H2O 0.01 g/L, FeSO4.7H2O 0.01 g/L, and NaCl 0.01 g/L. Unless else is mentioned, incubation and growth of the cultures was done in a shaking incubator at 37°C and 200 rpm.
Quantitative analysis of the volatile fatty acids (acetic and butyric acids) was done using GC (gas chromatography) (6890N, Agilent Technology, USA). Helium was used as the carrier gas. The oven temperature was programmed to increase from 50 to 240°C at a rate of 10°C/min. The temperature of the injection port and FID (Flame Ionization Detector) were 250°C. The concentration of the acids was determined according to a standard calibration curve. For analysis of the sugars and lactic acid, an HPLC (1200 Series, Agilent Technology, USA) with an RID (Refractive Index Detector) was used under the following conditions: mobile phase, 0.01M sulfuric acid; column, Aminex HPX-87H (dimensions - 300*7.8, Biorad, USA); flow rate, 0.5 ml/min; temperature, 40°C. The optical densities of the samples were measured using spectrophotometer (UV-mini 1240, Shimadzu, Japan) at 600 nm. To prove that [1, 2, 3 13C3] lactic acid is converted to butyric acid and not acetic acid by C. tyrobutyricum ATCC 25755T, spent culture broth was analyzed using GC equipped with a Pegasus 2D TOF-MS (LECO, St. Joseph, MI, USA) for detecting the presence of 13C in acetic and butyric acids. The column was HP-INNOWAX Polyethylene glycol (PEG) capillary column (17 m × 0.32 mm internal diameter, 0.25 μm film thickness, Agilent Technology, USA). The oven temperature was programmed to increase from 50°C to 150°C at a rate of 10°C/min. Helium was used as the carrier gas with a flow rate of 3 ml/min. The ionization mode employed was EI (electron impact ionization) with a scan rate of 100 spectra/sec and mass range of 40~500 m/z. The ion source temperature was 230°C .
Identification of the isolated Bacillus strain
The 16S rRNA gene sequence of the strain SGP1 was used as a query against the EzTaxon-e database [25, 26] and the 16S rRNA gene sequence of relatives were retrieved. The construction and evaluation of the phylogenetic trees were performed as described previously . The alignment and phylogenetic analysis were performed using MEGA5 . The 16s rRNA gene sequence of the strain SGP1 was submitted to GenBank (http://www.ncbi.nlm.nih.gov) and is registered under accession number HQ188291.1.
Fed-batch fermentations were done using a 3 L capacity bioreactor (Fermentec Co. Ltd., Korea) with an initial working volume of 1 L. The medium was RCM supplemented with 60.0 g/L sucrose (Sigma-Aldrich Co. USA). After autoclaving, the medium was purged with filtered oxygen-free argon gas. Overnight pre-cultures of the two strains were inoculated into the reactor at a concentration of 5% (v/v) for each strain. All fermentations were performed using agitation (200 rpm) and the pH was adjusted to the required values (5.3 – 5.9) by addition of a 10% ammonia solution, prepared by diluting an ammonium hydroxide solution (Cat # 320145, Sigma-Aldrich Co. USA) using sterile distilled water.
Observing levansucrase enzyme on an SDS-PAGE gel
A one-day (24 h) aerobic culture of B. sp. SGP1 was centrifuged and the supernatant was taken as a source for levansucrase enzyme. Concentration of the supernatant proteins was done through addition of acetone, centrifugation and then re-dissolving the precipitated proteins in phosphate buffer (pH 7.0). SDS-PAGE gel was then carried out as described by Laemmli with a 7% (w/v) acrylamide gel. After migration, the gel was cut into two halves. One half (containing the protein markers) was stained with Coomassie blue. The other half (containing the samples) was stained using periodic acid-Schiff reagent method [29–32] as follows: first, the gel was washed in a washing buffer containing 10 mM phosphate buffer (pH 7), CaCl2 (2 mM), and Tween 80 (10.0 g/L). Then the gel was incubated in the same solution but with 50 g/L sucrose, for 24 h at 37°C. After that, the formed levan was fixed on the gel through immersing in 70% ethanol, then the oxidizing solution (periodic acid o.7% (w/v), acetic acid 5% (w/v)) was added, followed by sodium bisulfate washing, and finally immersing in Schiff’s reagent (Sigma Aldrich, USA, Cat #; 3952016).
Sucrase and levan forming activity assay
The sucrase and levan forming activities at different temperatures and different pH values were determined by adding 50 μl of spent media, i.e. without cells, from a 20 h old B. sp. SGP1 culture to 950 μl of the reaction mixture (0.5 M sucrose solution in phosphate buffer) . To study the effect of the temperature, the pH was first adjusted to 6.6 before incubation at the test temperatures. Likewise, a temperature of 37°C was used when studying the effect of the pH. The incubation was performed for 10 h, after which, the sucrase and the levan forming activities were measured. Sucrase activity was determined by measuring the glucose liberated using glucose kits (Merck, Germany, Cat # 1.16720.0001), while levan formed was measured using the following procedure: 500 μl of the sample was added to 750 μl ethanol (to precipitate levan), and then the mixture was centrifuged. The precipitate was then washed with ethanol again, dried and then boiled with 0.1 N HCl for 30 min. Finally, the liberated fructose was analyzed using HPLC [14, 18].