Methods for producing high levels of carboxylic acids by lignocellulosic biomass processing
a technology of lignocellulosic biomass and carboxylic acids, which is applied in the direction of biofuels, microorganisms, biochemical equipment and processes, etc., can solve the problems of complex nutritional requirements, waste, and vast poorly exploited cellulosic biomass resources, and achieve high levels of acids
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example 1
Isolation and Cultivation
[0141]All procedures for enrichment and isolation of the strains listed in table 1 employed anaerobic technique for strictly anaerobic bacteria (Hungate 1969). The strains were enriched from environmental samples at temperatures higher than 70° C. with crystalline cellulose and beech wood as substrate. Isolation was performed by picking colonies grown on solid agar medium at 72° C. in Hungate roll tubes (Hungate 1969).
[0142]The cells are cultured under strictly anaerobic conditions applying the following medium:
Basic mediumNH4Cl1.0gNaCl0.5gMgSO4 × 7 H2O0.3gCaCl2 × 2 H2O0.05gNaHCO30.5gK2HPO41.5gKH2PO43.0gYeast extract (bacto, BD)0.5gCellobiose5.0gVitamins (see below)1.0mlTrace elements (see below)0.5mlResazurin1.0mgNa2S × 9 H2O0.75gDistilled water1000.0mlTrace elements stock solutionNiCl2 × 6H2O2gFeSO4 × 7H2O1gNH4Fe(III) citrate, brown, 21.5% Fe10gMnSO4 × H2O5gCoCl2 × 6H2O1gZnSO4 × 7H2O1gCuSO4 × 5H2O0.1gH3BO30.1gNa2MoO4 × 2H2O0.1gNa2SeO3 × 5H2O0.2gNa2WoO4 × 2...
example 2
HPLC
[0146]Sugars and fermentation products were quantified by HPLC-RI using a Via Hitachi LaChrom Elite (Hitachi corp.) fitted with an Rezex ROA Organic Acid H+ (Phenomenex). The analytes were separated isocratically with 2.5 mM H2S04 and at 65° C.
example 3
Phylogenetic Analysis of 16S rDNA Genes
[0147]Genomic DNA was isolated from cultures grown as described above and 16SrDNA amplified by PCR using 27F (AGAGTTTGATCMTGGCTCAG; SEQ ID No. 8) as forward and 1492R (GGTTACCTTGTTACGACTT; SEQ ID No. 9) as reverse primer. The resulting products were sequenced and the sequences analyzed using the Sequencher 4.10.1 software (Gene Codes Corporation). The NCBI database was used for BLAST procedures.
[0148]Sequencing of 16S rDNA from all strains listed in table 1 revealed all these had (at least) one copy of a 16S rDNA operon which was most closely related to Caldicellulosiruptor saccharolyticus (Strain Tp8T =DSM8903) in the available public databases. Alignment was carried out using ClustalW (Chenna et al. 2003) and the phylogenetic tree was constructed using software MEGA4 (Kumar et al. 2001). The tree for all strains listed in table 1 is displayed in FIG. 1.
[0149]The 16S rDNA sequences of all strains listed in table 1 have 99% percent identity to ...
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