Abstract
Standard microorganism isolating technology applied for complex multiphase environmental samples such as soil or sediment needs pre-treatment steps to remove living cells from their mixed-phase microniche, by creating a liquid-phase sample. This process removes synergetic relationships, which help to maintain viability of yet-to-be-cultured and hard-to-culture bacteria. In this paper we demonstrate a high throughput Laser Micro-Sampling (LMS) technology for direct isolation of pure microbial cultures and microbial consortia from soil. This technology is based on laser printing of soil microparticles by focusing near-infrared laser pulses on specially prepared samples of a soil/gel mixture spread onto a gold-coated glass plate. Microsamples of soil are printed on glucose-peptone-yeast agar plates, to estimate the LMS process influence on functional and taxonomic microbial diversity, and on «Eco-log» sole carbon sources microplates, to investigate functional diversity by “metabolic fingerprinting”. The obtained results are compared with traditionally treated soil samples. It was shown that LMS treatment leads to increasing of cultured biodiversity and modifies the functional diversity. The strain of rare genus Nonomuraea was isolated by LMS from complex natural environment without using media selective for this genus.Graphical abstract
Highlights
According to recent research, over 90% of bacteria from environmental samples remain uncultivable while using standard cultivation methods on trivial media [1,2,3]
Laser printing of soil microparticles Laser printing of simple water-soil mix results in severe spraying, uneven form, and large deviations in the diameter of the transferred droplets
A high throughput Laser Micro-Sampling (LMS) technology for direct isolation from soil of pure microbial cultures and microbial consortia has been developed. This technology is based on laser printing of soil microparticles by focusing near-infrared laser pulses on specially prepared samples of a soil/gel mixture spread onto a gold-coated glass plate
Summary
Over 90% of bacteria from environmental samples remain uncultivable while using standard cultivation methods on trivial media [1,2,3]. The variety of yet-to-be-cultured bacteria, especially the soil ones, seems to be a largest native prokaryotic gene pool on the Earth. It is remarkably significant for the phylogenetic studies and biotechnological research, in particular in context of new antibiotic producer findings [4]. The standard soil treatment procedures, such as an ultrasonic or vortex processing, imply dispersing and homogenization of a soil sample in liquids that assumes disintegration of soil microaggregates and particles, leads to desorption of microorganisms and Competition for resources between co-cultivated microorganisms, resulting in rapid development of dominant fast growing strains and in the lack of resources for slow growing microorganisms with chemical inhibition of their growth due to antibiotic production; In absence of growth on agar, application of synthetic media not fulfilling the growth requirements for a given organism and/or impossibility for to select proper artificial media for some highly associated groups, such as symbionts or pathogens; Breaking vital liaisons existing in the soil micro microzones peculiar to some organisms, served to sustain the regulation and metabolism.
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