Growth and maintenance of C. acetobutylicum
A frozen culture of C. acetobutylicum NCIM 2841 strain was procured from the National Collection of Industrial Microorganisms, India. Clostridial spores were inoculated in 100 Ml cooked meat medium containing the composition (g/L) of beef extract, 45; dextrose, 2.0; protease peptone, 20; NaCl, 5.0; and then incubated at 37 °C in an anaerobic condition. It was maintained in the cooked meat agar medium until the next use.
Preparation of anaerobic seed culture
The anaerobically digested slurry was collected from a functional biogas plant (3 m3) at the Biogas Research Centre, Gujarat Vidyapith, Sadra, India. It was filtered through a muslin cloth and centrifuged at 5000 rpm for 10 min by a bench top centrifuge. One volume of the collected supernatant was diluted with three volumes of dilution medium (g/L) containing NaHCO3, 5.0; NaCO3, 10; and two or three drops of resazurin (0.001%, w/v). It was pre-activated for 24 h at 37 °C before inoculation.
Enrichment technique for methanogens
Methanogens were enriched and cultivated by the anaerobic technique described previously (Wolfe 2011). Enrichment of methanogens was carried out in a medium containing the composition (g/L): NH4Cl, 1.0; NaCl, 0.6; NaHCO3, 5.0; KH2PO4, 0.3; K2HPO4, 0.3; MgCl2·6H2O, 0.16; CaCl2·2H2O, 0.009; and resazurin 0.001% solution, 1.0 mL. A solution of the following vitamins (10 mg/L): p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine, riboflavin, thiamine and 5 mg each of biotin, folic acid, α-lipoic acid, and cyanocobalamine B12 were prepared and 10 mL of this solution added to the above medium. A solution of trace minerals (g/L) was prepared and 10 mL of this solution mixed with 1 L of that medium: trisodium nitrilotriacetic acid, 1.5; Fe(NH4)2(SO4)2, 0.8; NaSeO3, 0.2; CoCl2·6H2O, 0.1; MnSO4·H2O, 0.1; Na2MoO4·2H2O, 0.1; NaWO4·2H2O, 0.1; ZnSO4·7H2O, 0.1; NiCl2·6H2O, 0.1; H3BO3, 0.01; and CuSO4·5H2O, 0.01. The pH of the medium was adjusted to 7.4 with 0.1 M KOH. Cysteine-HCl and Na2S·9H2O were added separately to this medium. After that, 100 mM sodium acetate for acetate-utilizing methanogens and 1% (w/v) gelatin for gelatin-enriched cultures were added separately in the enrichment media. The pre-activated seed culture (5%, v/v) was inoculated to initiate the enrichment of methanogens and then incubated at 37 °C under N2 headspace. Acetate was replenished everyday until the culture reached a late log phase.
Assay for specific methanogenic activity
Experiments were done in the anaerobic serum vials of 132 mL with a working volume of 50 mL phosphate-buffered basal (PBB) medium (Moench and Zeikus 1983). The PBB medium is composed (g/L) of KH2PO4, 1.5; K2HPO4·3H2O 2.9; MgCl2·6H2O, 0.2; CaCl2·2H2O, 0.1; NaCl, 0.9; NH4Cl, 1.0; resazurin, 0.001% solution, 1.0 ml; trace mineral solution, 10 mL; vitamin solution (µg/mL): biotin, 10; pantothenate (calcium), 25; lipoic acid, 25; folic acid, 10; thiamine, 25; riboflavin, 25; pyridoxine HCl, 50; cyanocobalamine B12, 0.05; nicotinic acid, 25 and p-amino benzoic acid, 25. Trace mineral solution has consisted of (g/L) trisodium nitrilotriacetic acid, 12.8; Na2SeO3, 0.02; CoCl2·6H2O, 0.16; Na2MoO4·2H2O, 0.01; H3BO3, 0.1; FeSO4, 0.1; MnCl2, 0.1; ZnCl2, 0.1; CuCl2, 0.02 and NiSO4, 2.6 mg.
After the proper enrichment, methanogenic culture (5%, v/v) was inoculated into 50 mL of the PBS medium containing 100 mM sodium acetate as an initial growth substrate. It was incubated at 37 °C until the methane production and depletion of acetate cease. After addition of 2–3 drops of titanium (III)-nitrilotriacetate reductant, the gas phase of a serum bottle was changed with N2 by evacuation and flushing. Sterilized gelatin at 1% (w/v) was added along with the overgrown culture of C. acetobutylicum and the time course was followed by incubation at 37 °C. A serum vial containing the same assay medium without gelatin was served as a negative control. The gas phase was replaced with N2 gas daily. Gas production was measured using an air-tight syringe. After the incubation, the entire content of the digested liquid was centrifuged at 5000 rpm for 15 min and the supernatant used for determination of pH, volatile fatty acids, and other metabolites.
Gas samples were collected from the head phase of each vial and immediately analyzed for methane content using a Gas chromatograph equipped with thermal conductivity detector (Perkin-Elmer Autosystem) as outlined previously (Chellapandi and Uma 2012a, b). One microlitre of diluted biogas sample was injected into a packed 2-m Porapak T steel column using an air-tight syringe. Nitrogen was used as a carrier gas at the flow rate of 30 mL/min. The oven, injection, and detector temperatures were adjusted to 50, 70, and 150 °C, respectively. Gelatin concentration was analyzed by using a modified Bradford dye-binding protein assay (Noble and Bailey 2009) and then calibrated with a gelatin standard. The concentration of extracellular and intracellular amino acids was calorimetrically determined with ninhydrin reagent by measuring optical density at 570 nm (Moore and Stein 1954) using glycine and alanine as standards. The liberated ammonia during gelatin degradation was estimated by spectrophotometer using sodium nitroprusside reagent at 625 nm and then calibrated with ammonium sulfate standard (Chaney and Marbach 1962).
The exchange metabolites of these cultures were detected from the digested fluid by GC–MS/MS (Perkin-Elmer Autosystem with turbo mass) with PE-5MS column (30 m; 250 micron internal diameter; 0.25 micron thickness) (Halket et al. 2005). Helium was used as a carrier gas. The oven temperature was adjusted to 80 °C for 5 min and raised to 280 °C with the rate of 10 °C/min and of 15-min hold time. Both injection and electron impact source temperatures were set at 250 °C. Split ratio was adjusted to be 1:60 and mass ranged between 20 and 650 atomic mass units. The volatile fatty acids and hydrocarbons were extracted in dichloromethane, concentrated, and then injected 1 µL sample for analysis. Metabolites present in the samples were identified from mass–ionization ratio obtained in GC–MS/MS peeks using the National Institute of Standards and Technology 11 and National Bureau of Standards Mass Spectral Library.