Enrichment cultures and isolation
Environmental samples, used for enrichment anaerobic cellulose degradation microflora, were collected from wheat straw compost, soil beneath wheat straw compost, rumen fluid, rumen solids, fresh cattle dung, rooted wood crumb, cattle dung compost, soil beneath cattle dung compost, and the activated sludge from wastewater digestion reactor. These samples were suspended in sterilized oxygen-free water with the ratio of solid and liquid 1:10 (w/v) and fiercely oscillated for 1 h followed by a static settlement for 10 min. Then, 10 mL supernatants were added into 90 mL enrichment medium, which contained (per liter) 10.0 g of filter paper, 1.0 g of (NH4)2SO4, 1.0 g of NaCl, 3.0 g of K2HPO4, 1.5 g of KH2PO4, 0.2 g of MgSO4, 0.5 g of CaCO3, 0.5 g of cysteines, and 0.2 mL trace elements solution . The pH value was adjusted to 6.5 with 5 M NaOH. Nitrogen (99.9 %) was used to form the anaerobic conditions. After 6-day fermentation with shaking (100 rpm) at 37 °C in an IS-RDH1 incubator shaker (Crastal Technology (Shenzhen) Co., Ltd.), 10 mL liquid cultures were then transferred to the brand-new enrichment medium for the second generation subculture. This process was repeated multiple times until the culture had a unique microbial community which was indicated by PCR-denaturing gradient gel electrophoresis (DGGE) analysis (see below).
To obtain cellulose-degrading isolates, the stable microbial consortium was diluted and plated on solid medium containing 10 g/L of microcrystalline cellulose (Avicel, PH101) instead of filter paper as sole carbon source, while the other components were the same with enrichment medium. Colonies surrounded by clear zones were subcultured to cellulose agar plates. Replicate plating was done several times to ensure the purity of the isolated colonies. Isolates with high butanol production potential from cellulose were identified and tested.
Total genomic DNA was extracted from enriched microbial consortium N3 using Bacteria DNA Mini Kit (Watson Biotechnologies, Inc., China) according to the manufacturer’s instructions. DNA extracts was used as a template for amplification of the 16S rRNA V3 fragment using Ex-Taq DNA polymerase (TAKARA Biotechnology Co., Ltd.). Primers used in this amplification process were BSF338/352 (5′-actcacccgtccgcca-3′) and BSR534/518 (5′-attaccgcggctgctgg-3′). The reactions were performed in a Peltier thermal cycler (Bio-Rad Laboratories, Inc.) with the conditions of initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s and extension at 72 °C for 1 min, and ended by a final extension at 72 °C for 10 min. The amplified 16S rRNA V3 fragments were analyzed by DGGE using the DCodet™ system (Bio-Rad) with a gradient concentration of denaturing agent ranging from 30 to 60 % in polyacrylamide gel. The DNA fragments recovered from the gel were used as templates for re-amplification under the reaction conditions described above, and the resulting PCR products were cloned for sequencing.
16S rRNA gene sequencing
The isolate genome DNA was extracted and used as templates for PCR amplification with primes BSF8/27 (5′-agagtttgatcctggctcag-3′) and BSR1492/1474 (5′-ggttaccttgttacgactt-3′). Amplification was performed in a 9700 PCR meter (Bio-Rad Laboratories, Hercules, USA) with the conditions as mentioned by Chen . The sequences were initially compared to the available databases using the BLASTn sever to determine their approximate phylogeny . A phylogenetic tree was constructed using the neighbor-joining method provided in MEGA version 5.1 .
Batch fermentation tests
Fermentation studies were conducted in oxygen-free medium which contained the same components with enrichment medium except for 30 g/L filter paper cellulose. In the co-culture system, a volume of 2 mL microbial consortium N3 or isolate C. celevecrescens N3-2 in their exponential-growth phase was added into 96 mL fermentation medium, after 48 h fermentation, another volume of 2 mL C. acetobutylicum ATCC824, a butanol-producing strain purchased from China General Micro-biological Culture Collection Center (CGMCC), also in its exponential-growth phase, was added in.
In all cases, the fermentation was carried out at 37 °C and 100 rpm, with a final reaction volume of 100 mL. During fermentation, samples were taken at predetermined intervals. To check data reproducibility, triplicate sets were carried out in each experiment.
Liquid products of fermentation (ethanol, acetone, butanol, acetate, and butyrate) were analyzed by using gas chromatography (GC; 6890N, Agilent Technologies, Santa Clara, CA), and gases were measured by a thermal conductivity detector after separation by GC (GCSC2) (Shanghai Analytical Apparatus, Shanghai, China) as described by our previous study . Sugar concentration during fermentation was determined using a high-performance liquid chromatography (HPLC) system (LC-10A, Shimadzu Corp., Kyoto, Japan) following the method reported by Cao . Cell mass was determined indirectly by measuring the total protein . Briefly, the fermentation broth was firstly disintegrated by ultrasonication, then the supernatant was collected after centrifugation at 12,000× g for 5 min and tested with Coomassie brilliant blue (CBB) by spectrophotometric analysis at 595 nm according to the method of Bradford. For residue cellulose analysis, the fermentation broths containing cell mass and cellulose were centrifuged at 12,000× g for 15 min to separate the supernatant and precipitate. Then, the precipitate was determined gravimetrically after drying at 80 °C for 2 days with non-inoculated medium as a control. After that, the residue cellulose was calculated by subtracting the amount of cell mass which was determined indirectly by measuring the total protein. The activities of endoglucanase, exoglucanase, and β-glucosidase were determined using the supernatant of the fermentation broth after a centrifugation at 12,000× g. The substrates for each enzyme were CMC-Na, microcrystalline cellulose, and salicin, respectively. The reactions were carried out at the temperature of 55 °C in pH 6.0 for 30 min according to the method reported by Cao . Then, the reducing sugar was measured and calculated into enzyme activities. One unit of enzyme activity (IU) was defined as the amount of enzyme which produced 1 μmol of reducing sugar per 1 min. For carbon balance calculations, the elemental composition of the microbes was assumed to be C5H7NO2 . Electron balances were calculated a ratio of oxidized products to reduced products (O/R ratio) as a function of the available electrons per mole of the substrate and products [28, 29]. To check data reproducibility, triplicate sets were carried out in each experiment.