Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target

Abstract

By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.

Expression of different types of [FeFe]-hydrogenase genes in bacteria isolated from a population of a bio-hydrogen pilot-scale plant

“…So far, the high diversity of [FeFe]-hydrogenases has been studied both in natural environments [25][26][27] and several pilot scale plants for H2 production [28][29][30].…”

Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

“…Thus far, however, there have been rare successful applications of FISH-flow cytometry approaches targeting mRNA [26,29,30].…”

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

“…As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination 1,2 , cellular identification and gene expression 3,4 , monitoring viral multiplication in infected cells Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T m ) of these analogs for natural nucleic acids 7,8 .…”

“…, cellular identification and gene expression 3,4 , monitoring viral multiplication in infected cells 5 , and bacterial community analysis and enumeration 6 .…”