Fondamenti di microbiologia applicata - Dr. Laura Treu Dipartimento di Biologia E-mail: e-learning unipd
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Fondamenti di microbiologia applicata
Dr. Laura Treu
Dipartimento di Biologia
E-mail: laura.treu@unipd.it
Istituto Vallisneri, Viale G. Colombo 3
Laboratorio 16, terzo piano sud,
Telefono 049 827 6306
@DiBio_UniPD
A. A. 2018-2019
Fondamenti di microbiologia
We are immersed in microbes. They live in our bodies, in our food, and
in everything that surrounds us.
Microbiology is the study of living things too small to be seen without
magnification.
Microbes or microorganisms are microscopic organisms smaller than 0.1mm,
characterized by activities typical of biological systems.
Microbes are related to all aspects of life:
• in all environments
• many beneficial aspects
• related to life processes (food, nutrient cycling)
• only a minority are pathogenic
A. A. 2018-2019
A. A. 2018-2019
Eucarioti vs Procarioti
• Fungi composed of molds (multicellular) and
yeasts (unicellular) • Bacteria single-celled
• Animals multicellular • Archaea single-celled
• Plants multicellular
• Protozoa single-celled
• Algae unicellular or multicellular, photosynthetic
A. A. 2018-2019
5
Eucarioti vs Procarioti
Procarioti
Semplici organismi unicellulari,
generalmente privi di membrane
endocellulari e organelli. Si
riproducono principalemnte attraverso
scissione binaria. Il genoma (DNA
circolare) si colloca libero nel
citoplasma.
Eucarioti
Organismi che presentano un nucleo,
dove è contenuto il DNA, e con dei
compartimenti interni racchiusi da
membrane, gli organelli, che
svolgono particolari compiti biologici.
Alcuni di questi organelli presentano
uno specifico genoma.
A. A. 2018-2019
La crescita microbica
In microbiology growth is defined as an increase in number of cells.
In prokaryotes one cell divides into two new cells (binary fission).
One
generation
https://www.youtube.com/
watch?v=gEwzDydciWc
A. A. 2018-2019
Il tempo di generazione
Generation time is dependent on temperature, nutrients, substrates, genetic
etc.
Some environmental factors affecting microbial growth are:
• Temperature
• pH
• Oxygen concentration
E. coli has a generation time of 20 min., but most bacteria and archaea grow slower.
A. A. 2018-2019
La crescita esponenziale
Example:
• After 20 hours 1 gram of bacteria has been produced.
• After 31 hours app. 4 tons of bacteria has been produced (= 1 elephant)
• After 42.5 hours an amount of the entire human population has been produced.
A. A. 2018-2019
Le fasi di crescita
• Lag phase: Microorganisms need to adapt to growth conditions.
• Exponential phase: no limitations in growth conditions (nutrient etc.).
• Stationary phase: no net production in cells, but metabolism may continue.
Nutrient are limited or waste product may inhibit the microorganisms.
• Death phase: Cells die and cell lysis might occur.
A. A. 2018-2019
I biocatalizzatori
Biocatalyst: enzyme, cell, or a group of enzymes or
cells catalyzing a chemical reaction or series of
chemical reactions.
Use of enzymes produced separately by
microorganisms
• Harvest/purify enzymes and use in
applications
• Substrate too big to enter the cell (e.g.
degradation of cellulose to glucose)
Use the biochemical activity of the whole cells
• Example : Fermentation of ethanol from
glucose
A. A. 2018-2019Tipologie di relazioni
Symbiotic describes an intimate relationship between two organisms.
There are three types of symbiotic relationships:
• Mutualism - both benefit
• Commensalism - one benefits and the other receives no benefit,
but is not harmed
• Parasitism - one benefits and the other is harmed
Rhizobium Staphylococcus aureus
A. A. 2018-2019Ecologia microbica
Biodiversity: isolate, identify and quantify the microbes.
• Morphological techniques: what do they look like?
• Culture based techniques: what do they grown on?
• Molecular techniques: who are they related to?
Microbial activity: measure what the microbes are doing.
• Physiological techniques: physiological properties?
• Molecular techniques: what they can do?
What is the microbial biomass of a biogas reactor? Or any other complex environment (soil,
marine sediment, biofilm etc.) ..and what do we want to know?
A. A. 2018-2019Microscopia Morphological technique
The two basic microscopes are:
• the light (optical) microscope uses visible light to cell stuctures. Light
microscopes are used to look at intact cells at low magnification. Types: bright-
field, phase-contrast, dark-field, fluorescence and Confocal Scanning Laser
Microscope (CSLM - 3D imaging)
• the electron microscope uses electrons and can
be used to examine internal structures of
microorganisms at high magnification.
Types: Scanning Electron Microscopy
(SEM) and Transmission electron microscopy
A. A. 2018-2019Tecniche di colorazione
Methylene blue, crystal violet. Can increase contrast
of a simple
bright-field light microscope
Gram-staining. Gram-positive cells appears blue and
gram negative cells appears
red.
DAPI is a widely used fluorescent dye staining cells
bright blue. The cells can be
identified with a fluorescence microscope in an even
complex milieu.
Streptococcus pneumonia
biofilm
A. A. 2018-2019Microscopia elettronica
A. A. 2018-2019Isolamento, crescita e quantificazione
• Spread plating: isolation and quantification of cells able to form colonies on
solid media (agar) that may include compound to enrich or restrict growth of
specific microbes.
• Streak plating: isolation of single bacterial colony.
• Counting chamber and Turbidity: quantification of cells growing in liquid
media.
Advantages: rather simple and fast methods
Disadvantages: the microbes need to be able to grow on/in the medium.
A. A. 2018-2019Streak plate
Dilution and isolation of bacteria with inoculation loop or needle on petri plates.
A. A. 2018-2019Tipologie di mezzi dicrescita
Synthetic of Defined Media: usually relatively simple media, all components are
known.
Complex Media: composition of media not completely known. Often made from
inexpensive organic materials such as slaughterhouse wastes (tryptic digests called
tryptone, trypticase, etc.), soybeans, yeast wastes from brewing (rich source of
vitamins), animal blood, etc.
Selective Media: media favors the growth of one or more microbes.
Example: bile salts inhibit growth of most gram-positive bacteria and some gram-
negative bacteria, but enteric bacteria adapted to life in animal gut can grow well.
Differential Media: media allows distinguishing between different bacteria that grow.
Example: MacConkey agar has color indicator that distinguishes presence of acid.
Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce
acid wastes on plates, turn pH indicator red. Bacteria that cannot ferment the same
sugar will grow but not affect pH, so colonies remain white.
A. A. 2018-2019Esempi
Unknown micro-organisms (e.g. environmental)
• Cultures on Petri plates using
complex medium
• Colony characterization by
morphology
Disadvantage: total cell count of a sample using a single medium will always
give a clear underestimation of the real number of cells.
Well known micro-organisms (e.g. pathogens)
• Cultures on Petri plates using specific
and selective medium
Advantage: selective media can yield rather reliable data related to specific microbial
physiological properties.Camere di conteggio
Total cell count with Microscope
Advantage: very fast procedure
Limitations:
• dead cells are not distinguished from living cells
• small cells are difficult to see
• motile cells must be immobilised
A. A. 2018-2019Torbidità
Measurement on spectrophotometer of unscattered light in a cell suspension,
optical density (OD).
A spectrometer works by splitting light made
of many wavelengths (e.g. white light) into
individual rays that can be detected.
This allows the spectrometer to
be able to find the absorbance of
different wavelengths and
determine what molecules are in
the solution.
A. A. 2018-201922
Bacteria Archaea Yeast
Methanosarcina Methanobacteriales
Bacillus + Clostridium S. cerevisiae
Thermoanaerobacter Methanosaeta
A. A. 2018-2019L’informazione genetica: il DNA
Approx. 1 million genes
Approx. 3000 to 6000 genes
All living organisms contain DNA, it encodes all of the information needed to program the
cell's activities (reproduction, metabolism and specialized functions). All of the DNA found in
an organism is collectively referred to as the genome.
A. A. 2018-2019Next Generation Sequencing
Next Generation Sequencing (NGS), also known as high-throughput
sequencing, is the catch-all term used to describe a number of
different modern sequencing technologies:
Illumina (Solexa)
Roche 454
Pacific Biosciences
Before 1998 Sanger sequencing provided only one single sequence
at time with very high costs
With NGS can be obtained up to 4 billion reads, i.e 4000000000, in a
single sequencing run
NGS is significantly cheaper, quicker, needs significantly less DNA
and is more accurate and reliable than Sanger sequencing.
More info:
https://www.ebi.ac.uk/training/online/course/ebi-next-generation-sequencing-practical-
course/what-you-will-learn/what-next-generation-dna-
A. A. 2018-201925
Alcuni numeri della biologia
Guerra e pace: 3 millioni di Il genoma umano: 3 miliardi di
lettere basi
Oshlack, A., Genome Biology 2013, 14:104.
Informatica e Bioinformatica – A. A. 2018-201926
Gli organismi "inaccessibili"
Microbes that grow readily in culture
represent only a fraction of the living
organisms of interest
Many species are difficult to study in
isolation because they fail to grow in
laboratory culture, depend on other
organisms for surviving
Methods that are based on DNA
sequencing circumvent these
obstacles, as DNA can be isolated
directly from living cells in various
contexts
Such methods have led to the
emergence of a new field, which is
referred to as METAGENOMICS
Tringe S.G., 2005 Nature Reviews
A. A. 2018-2019Approcci genetici
Fluorescence In Situ Hybridization (FISH): single microorganisms
identification
P Quantitative PCR (rt-PCR, qPCR): specific genes are targeted
C
R 16S High Throughput Sequencing: several 100 of rRNA 16S gene sequences
Total random sequencing (TRS): metagenomics, millions of sequences
RNA-seq: metatranscriptomic, whole community gene expression
A. A. 2018-2019Metagenomica e metatranscrittomica
Up to 100 samples for run
Phylogenetic classification
16S rRNA Dominant and low abundant
microorganisms
gene
Phylogenetic classification Phylogenetic classification and
and gene functional metabolic activity
annotation Gene expression for microbial
Up to 10 metagenomes functional characterization
in a single run
Total Random
mRNA seq
Sequencing
NGS for disclosing microbial diversity (2014)
A. A. 2018-201916S ribosomal RNA gene
Uncultivated species are identified by their 16S/18S small subunit
ribosomal RNA (SSU rRNA) genes
These genes are commonly used as phylogenetic markers because
every cellular organism contains them and almost all gene variants can
be amplified by standard sets of degenerate universal primers
16S ribosomal RNA (rRNA) sequence can provide a unique molecular
‘bar code’ for identifying an organism and placing it in an evolutionary
context
Comparison of these sequences with databases of known 16S
ribosomal RNA genes allows them to be phylogenetically classified
Estimation of the community structure is provided, as sequences from
dominant community members are more abundant.
Tools (e.g. ARB and CLC) and databases (e.g. Ribosomal Database
Project) have been developed to manage and analyse this flood of data.
Tringe S.G., 2005 Nature Reviews
A. A. 2018-201930
rRNA e tassonomia
Nella sequenza del 16S rRNA dei procarioti
si riconoscono delle regioni conservate
intervallate a regioni ipervariabili.
Tramite metodiche di biologia molecolare
(reazione di PCR e sequenziamento) si
riesce ad analizzare specificatamente questo
gene.
Con tassonomia si intende lo studio delle
relazioni filogenetiche tra organismi diversi.
La conoscenza della sequenza delle regioni
ipervariabili consente di assegnare la
tassonomia di un dato organismo.
A. A. 2018-2019Tassonomia e nomenclatura
Taxonomy is the study of
phylogenetic relationships
between organisms. It’s based on
hierarchical classification system
and the binomial nomenclature.
A. A. 2018-201916S rRNA conserved regions
A. A. 2018-201933
Polymerase Chain Reaction (PCR)
La Polymerase Chain Reaction è una tecnica per l’amplificazione “in vitro” di
specifiche sequenze di DNA, attraverso la simultanea estensione di filamenti
complementari di DNA. Il metodo fu inventato da Kary Mullis nel 1983 e gli valse
un premio Nobel per la chimica nel 1995.
A. A. 2018-201934 A. A. 2018-2019
A. A. 2018-2019
Fluorescence In Situ Hybridization (FISH)
Epifluorescent microscope :
Red cells = E. coli vs Green cells = Clostridium sp.
A. A. 2018-2019Identificazioine di nuove specie microbiche
Genome size [bp] 2.15 Mbp
Number of contigs 503 Candidatus Methanoculleus
Number of protein-encoding genes 2,297 thermohydrogenotrophicum
Estimated completeness % (CheckM) 92.70%
Kougias P.G. et al. 2017 Anaerobe
A. A. 2018-2019Metagenomica
Established the first Progressive functional
reference catalog of specialization leads
biogas microbial to the final step of the
genomes (2016) process
Campanaro, S., ...Angelidaki, I. 2016. Biotechnology for Biofuels
A. A. 2018-2019Mesophilic community Thermophilic community
Coverages changes before (light) and after (dark) H2 addition are represented as circles
with proportional areas
Treu L. et al. 2018 Frontiers in Microbiology
A. A. 2018-201941 Metatranscrittomica Methanogenic pathway reconstructions in archaea and the expression levels of relevant genes. CoA, coenzymeA; MFR, methanofuran; H4MPT, tetrahydrosarcinapterin; HS-CoM, coenzyme M; HS-CoB, coenzyme B; MP, methanophernazine A. A. 2018-2019
42 Domande...? A. A. 2018-2019
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