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

2. 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)

3. Spallanzani - 18th century
The early attempt
Spallanzani - 18th century
L.Pasteur th 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.

4. 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. )

5. 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.”

6. 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

7. 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

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

9. 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

10. 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