The duo Clostridium and Lactobacillus linked to hydrogen production from a lignocellulosic substrate.

The study aimed to identify interspecies interactions within a native microbial community present in a hydrogen-producing bioreactor fed with two wheat straw cultivars. The relationships between the microbial community members were studied building a canonical correspondence analysis and corroborated through in vitro assays. The results showed that the bioreactor reached a stable hydrogen production of ca. 86 mL/kg·d in which the cultivar change did not affect the average performance. Lactobacillus and Clostridium dominated throughout the whole operation period where butyric acid was the main metabolite. A canonical correspondence analysis correlated positively Lactobacillus with hydrogen productivity and hydrogen-producing bacteria like Clostridium and Ruminococaceae. Agar diffusion testing of isolated strains confirmed that Lactobacillus inhibited the growth of Enterococcus, but not of Clostridium. We suggest that the positive interaction between Lactobacillus and Clostridium is generated by a division of labor for degrading the lignocellulosic substrate in which Lactobacillus produces lactic acid from the sugar fermentation while Clostridium quickly uses this lactic acid to produce hydrogen and butyric acid. The significance of this work lies in the fact that different methodological approaches confirm a positive association in the duo Lactobacillus-Clostridium in a bioreactor with stable hydrogen production from a complex substrate.

Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74.

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine-Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima).

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5alphaF', and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55 degrees C.