Microbes in Nature I: Diversity & Occurrence
Siu-Wai CHIU
Department of Biology & Environmental Science Programme
Micro-organisms exist in every niche of our biosphere. Micro-organisms have been
applied in various aspects: fermentation for production of pharmaceuticals, food and
food products, biocontrol, genetic engineering, bioremediation, biomining, etc.
However, what defines a micro-organism and how diverse micro-organisms are. These
will be discussed in the following sequence:
Outline
1. What are micro-organisms?
2. What are the differentiating features among the major groups of
micro-organisms
3. Diversity & occurrence:
A.Virus as the most successful parasite:
a. life & non-life,
b. minimum size of genome but
c. the rapid generation of natural genetic variants by genetic recombination
d. its territory is defined by its host.
B. Fungi are commonly employed in fermentation for production of useful
biomasses and metabolites, e.g. alcohol, mushrooms, enzymes.
a. active decomposers in recycling of nutrients
b. Over 90% plant roots have one or more fungi as mycorrhizae helping the
uptake and absorption of nutrients.
c. pathogenic fungi cause great loss in crop production but mild as mycoses
in man
d. rumen fungi indicate that fungi can lead anaerobic life.
e. Some regard fungi so distinct that they should form a kingdom of their
own. Briefly, their unique features include: metabolism is more
animal-like, but mostly sessile and have cell wall although the cell wall
composition is rarely cellulose and these fungi never carry out
photosynthesis.
C. Algae are the predominant primary producers capturing solar energy and
fixing atmospheric carbon dioxide.
a. some algae can carry out heterotrophic mode of nutrition to decompose
excess nutrients in open water causing algal bloom. Red tide is a specific
form of algal bloom.
b. Thus algae inhabit areas usually with light and with oxygen. Cysts,
however, are the forms surviving adverse conditions such as the dark
ocean sediment and the icy floor where water becomes unavailable in the
format of solid.
c. Lichen is a symbiosis situation enabling unicellular algae to invade dry
and exposed terrestrial habitats
d. Subgroups of algae are differentiated by the number and form of flagella,
photosynthetic pigments, cell wall composition and storage products.
Morphology can help but not the only criteria for classification.
D. Bacteria: the most diverse prokaryotes in terms of
a. morphology, structure, ecological roles, life requirements and habitats.
For instance, the parasitic Mycoplasma, a human pathogen, does not have
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a cell wall. Rod-shaped and flagellated Escherichia coli is facultative
anaerobic. Spherical Deinococcus radiodurans equipped with a powerful and
efficient DNA repair machinery can withstand radiation of 1.5 million rads of
ionization radion, 3,000 times what it would take to kill a human.
b. Blue-green bacteria: originally named as blue-green algae as these do
carry out photosynthesis, a property shared with algae.
c. Actinomycetes commonly found in soil and are applied for fermentative
production of bioactive compounds including antibiotics, enzymes and
vitamins. They have long been regarded as fungi since this group of
organisms are filamentous and carry out sporulation. Common keys for
pathogenic filamentous fungi still include actinomycetes as they are so
similar in morphology except for the smaller size in actinomycetes.
d. Escherichia coli is a well-known microbe. It is a normal resident of the
large intestine. Thus, large population size in an aquatic body suggests
fecal contamination. With its fast growth (doubling time within 20 min),
ease to cultivate in laboratory condition and low pathogenicity, it is used
as a model bacterium for studying the biological science of bacteria, e.g.
mating. With artificial manipulation, E. coli is commonly used as a
cloning host in genetic engineering. However, virulent strains can be
lethal, e.g. strain O157: H7 produces a toxin causing kidney failure and
haemolysis in intestine.
Case studies:
1. Nano-bacteria: isolated from Triassic and Jurassic sandstones;
cell dimension: 20 – 150 nm; which is much smaller than most
bacteria
Genome: positive staining with DNA- specific dye
Grow aerobically. I.e. viable isolation
Transmission electron micrographs: possess an outer layer or
membrane representing a wall layer; ; naked nucleic acids
Scanning electron micrographs: filamentous with suspectible
sporulation structure
2. Was there life on Mars?
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Microbes in Nature II: Isolation, Detection & Identification
Siu-Wai CHIU
Department of Biology & Environmental Science Programme
Although micro-organisms live in every niche of the biosphere far beyond the boundary
of human inhabitation, 95-99% of these micro-organisms cannot be successfully
isolated nor cultured. Such failure is because of our deficiency in knowledge of the
microbial world, the complexity of its required environment and the difficulty in setting
up the required environment. In addition many organisms usually live and interact
together in nature. Thus isolation and sampling of a particular micro-organism may
very much reflect a density-dependent probability. Thus, isolation is not a simple
routine procedure but a challenging strategy.
For a micro-organism to grow, environmental conditions must be favourable. For
instance, the Chinese straw mushroom (Volvariella volvacea) does not survive at 4C,
and the black oak mushroom (shiang-gu, shiitake, Lentinula edodes) does not survive at
or above 32C. Thus, incubation temperature is critical. Another parameter needs to be
regulated is pH. Yeast Candida albicans is an opportunistic pathogen inhabiting in the
acidic vagina where most bacteria are not favoured to grow. Isolation in aerobic
condition kills the rumen fungi and many obligate anaerobes. The essential nutrients
required by most micro-organisms include C, N, P, O, S and H. Other minerals needed
include: K, Mg, Ca, Fe, Cu, Zn, Co and Mn. Some require complex growth factors
and/or vitamins, and thus complex ingredients such as yeast extract, potato extract,
blood agar or bovine serum are included in the cultivation medium.
Community studies are commonly found in the literature but the present culturing
based assays are inherently biased. Any organisms which are unable to survive in the
culture conditions will not be noted and recorded. Any slow growers exceeding the
isolation experimental period will be neglected. Also, because isolation can be biased,
this isolation strategy can be applied as selection, i. e. only select the target organism
with a certain phenotype. For instance, isolation of pollutant-degrading
micro-organisms is of biotechnological interest. To do this, the target pollutant is added
to the cultivation medium and/or isolation of micro-organisms from the
pollutant-contaminated site. The former strategy employs the pollutant as a selective
force. The latter employs the principle of ‘survival of the fittest’; if the micro-organism
does not adapt to the polluted environment by tolerance or degradation of the pollutant,
it will not be there. Screening from the isolated micro-organisms for a potential
pollutant degrader sometimes require complex biochemical procedures to elucidate the
degradation pathway or analyzing the breakdown products/intermediates using
advanced instruments.
In the lecture, the principle of an isolation and purification procedure for an edible
mushroom species is introduced. Students will practise the procedure during the
laboratory session. In brief, when started with a collection from the field or a live
commercial mushroom product is provided. Perform all the procedure with aseptic
techniques. Surface sterilization by wiping with 75% alcohol helps to remove some
airborne microbial contaminants. Then access to the inner unexposed tissues by cutting
the mushroom with a flame-sterilized knife and then followed by splitting open the
mushroom tissue. Aseptically cut and pick up the inner tissue with another sterilized
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knife or forceps. Then inoculate the excised tissues onto a nutrient-rich medium with
antibacterial antibiotics (the mushroom is a fungus. Antibacterial antibiotics is only
effective against bacteria found in the mushroom tissue and from contamination as a
result of improper procedure, e.g. airborne contamination, inoculation tools are not
sterilized, contamination from unclean operator’s hands). Then enrichment of the
isolated culture is needed to specifically mass produce the isolate. This purified isolate
can be used for fermentative production of microbial metabolites and biomass such as
cellulase by fungus Trichoderma, mushroom production, proteinase by fungus
Rhizopus, penicillin by fungus Penicillium, antibiotic streptomycin by bacterium
Streptomyces. In the laboratory session, students will get hold of the experience in
fermentative production of an oyster mushroom Pleurotus pulmonarius.
Identification is an artificial method relying on our knowledge towards
micro-organisms. For instance, lingzhi is a fungus of medicinal properties. Its
distinctive features only appear in the sexual reproductive stage. Both macroscopic and
microscopic features are needed for identification. As for bacteria, a differential
staining procedure will be needed to distinguish gram (+) and gram (-) bacteria whose
cell wall composition and structures are different. As many rumen fungi are obligate
anaerobes, artificial cultivation was not feasible in the 70s and 80s. Its true identity was
reflected by a specific staining reaction revealing the presence of chitin in its cell wall.
In bacteria, phospholipid fatty acid profile is commonly used, and is based on the
extraction of ‘signature’ lipid biomarkers from the cell membranes and walls. Analysis
of the component fatty acids of these extracted lipids is achieved by gas
chromatograph-mass spectrometer. Sulphate-reducing Desulfovibrio can be
distinguished from sulphate-reducing Desulfobacter by this method. Thus,
chemotaxonomy is very commonly applied for micro-organisms. It circumvents the
situation where many groups of micro-organisms are similar in morphology, and the
essential feature for identification only appears at certain life phase. In bacteria
identification and now exploited to yeast identification, a numerical way of matching
the substrate-utilization abilities of a unknown organism with those of the reference
species and then followed by a similarity measure or probability measure is in practice
e.g. Biolog microstation. With the advancement and convenience of molecular
techniques, polymerase chain reaction, DNA hybridization and DNA sequencing can
help identify all forms of organisms. Polymerase chain reaction is an in vitro
amplification of DNA using a thermostable DNA polymerase. Hybridization is based
on the nucleotide complementarity between a target DNA sequence and a
reporter-labeled probe. DNA sequence can yield phylogenetic information, population
diversity measure and is applied for identification.
As culturing based methods are inherently biased, and sometimes the purpose of the
study is to realize the presence of a particular organism in an environment instead of
isolation, then the alternative molecular based methods provide the whole picture by
measuring 100% of the microbes present. Such an approach yields information on
activity, metabolic status and an identification of the specific organism present in the
sample. For instance, specific DNA probes are available to detect for petroleum
degradation, i.e. to assess whether the gene encoding a specific enzyme for degrading
petroleum is present in the sample, e.g. nah H probe for detecting PAH degradation
ability. Also, a genus-specific or group-specific probe is also available, e.g. amo A for
detecting nitrifying bacteria.
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