Unculturable Bacteria Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar, Gujarat

Unculturable Bacteria Life on earth Thought to be ~3.8 billion years old. For the first 1.5 billion years it was all aquatic microbes. Less then 2% bacteria are presently cultured in the laboratory (an estimate)

Spallanzani - 18th century The early attempt Spallanzani - 18th century L.Pasteur - 19th century Were the first to use meat broth or similar infusion for bacterial culture. However, the drawback was that the broth contains mixture of bacteria.

Robert Koch Was the first to use potato slices as a solid surface to give colonies on its surface. However, he soon realize that only a limited number of organism grew on potato. This was probably the first recognition of unculturability “In vitro”. “The great plate count Anomaly” Size and diversity (8,00,000insect spp is known and 7000 bacterial spp. )

What is Unculturability? “Unculturability means the organisms are unable to grow in laboratory otherwise these organisms are very well growing rather flourishing in one or other natural environment.”

Reasons for Unculturability “In Vitro” High nutrient content Absence of required nutrient Toxic substance produced by other bacteria Some bacteria may depend on other for growth Environmental conditions are not suitable Disruption of bacterial cytokine networks

Cultivating Unculturables-Early attempts Enriched and Enrichment cultivation Media with low nutrients Modifying the cultural conditions:- Carbon dioxide and oxygen conc. Long incubation Addition of humic acid etc. Simulation of natural environment Gel micro droplets

Culture independent approach Metagenomics Various environmental samples were used to construct metagenomic libraries Small inserts with 2-15 Kb in plasmid vector Large inserts with40-130 Kb in cosmid, fosmid or Bacterial Artificial Chromosome (BAC) vector

Metagenomic DNA Plasmid library Cosmid or BAC library Activity or sequence based screening Single gene product- Biocatalyst Multiple gene product- Bioactive compound Sequencing and characterization

Biocatalyst and Bioactive compound isolated from metagenomic libraries Product Vector Sample Agarase Cosmid Soil Alcohol oxidoreductase Plasmid Soil Amidase Cosmid Soil Amylase Cosmid,BAC Soil Cellulase Cosmid Soil Chitinase Lambda-ZAP Marine water DNase BAC Soil Esterase Plasmid Soil Lipase Plasmid Soil Indirubin BAC Soil Turbomycin BAC Soil Violacein Cosmid Soil