M.Sc.

SEMESTER - III
MICROBIAL DIVERSITY
(Code: MBM - 301)
Unit-3
Extremophiles - acidophilic, alkalophilic, thermophilic, barophilic and osmophilic microbes -
mechanisms and adaptation. Halophiles - membrane - variation - electron transport -
application of thermophiles and extremophiles.

- Dr. Parvez Ahmad

High Sugar Concentration - Osmophiles

Osmophiles are the microorganisms that live in environments with high osmotic pressure.

They can live in environments with low water activity such as high sugar concentrations.

Although, these high sugar concentrations serve as a growth-limiting factor to many

microorganisms.

However, osmophiles, mainly yeasts and some bacteria can protect their cell against high
osmotic pressure generated by these high solute concentrations.

Moreover, they synthesize osmoprotectants including alcohols, sugars, amino acids,

polyols, betaines, and ectoine as an adaptation to these environments.

Generally, osmoprotectants compatible solutes are either neutral or zwitterionic.

They act as osmolytes, which maintain fluid balance and cell volume. Additionally, plants
also accumulate osmoprotectants during drought periods.
Osmophilic yeasts can cause spoilage of honey, chocolate candy with soft centers, jams,
molasses, corn syrup, flavored syrups and toppings, concentrated fruit juices, and similar
products.
Many of the common spoilage yeasts in this group belong to the genus
Zygosaccharomyces.

Figure: Saccharomyces cerevisiae, an Osmophile

Osmophiles are similar to halophillic (salt-loving) organisms because a critical aspect of
both types of environment is their low water activity, aw.
Nearly all osmophillic microorganisms fall under the yeast genus.
Many microorganisms are exposed to water stress and have developed methods of dealing
with it, but relatively few have evolved the physiological adaptations that enable them to
grow well in environments with low availability of water.
water activity = water availability
A low water activity environment has a value of 0.85 or less, and microorganisms that
grow well in such conditions have been described variously as: halophilic, osmophilic,
osmotolerant, etc.
Xerophilic fungi are yeasts and moulds that are capable of growth at or below a water
activity (aw) of 0.85.
These microorganisms have developed physiological mechanisms that enable their
biochemical pathways to function in environments where little water is available.
External conditions of low aw are sensed by membrane osmosensors, and xerophiles then
accumulate glycerol as a compatible solute to balance the internal and external osmotic
pressure.
They also modify their membranes to retain this glycerol within the cell.
There are a number of proposed strategies that are employed by xerophilic fungi to enable
them to survive and grow at reduced aw.

Those can vary in different fungal groups, but the main principles appear to be similar.
The most well-studied response to increased osmotic pressure is the intracellular
accumulation of a solute or solutes to maintain a balance between the aw inside the cell
and the external aw, thus preventing movement of water out of the cell.

At low aw, these solutes must be accumulated to very high concentrations, but they must
not inhibit the cell’s normal functions, such as protein synthesis or deoxyribonucleic acid
(DNA) replication.
Thus, they are known as compatible solutes.
One of the most well-studied compatible solutes in fungi is glycerol.
Glycerol as a compatible solute

It has been known since 1945 that xerophilic (osmophilic) yeasts produced polyhydric
alcohols, particularly glycerol, from sugar fermentation, but it was not until 1972 that
Brown and Simpson proposed a physiological function for this accumulation of polyols in
these yeasts.

They concluded that the primary function of the polyols (mainly arabitol and glycerol)
was to act as compatible solutes, enabling the vital processes of the cell to be maintained,
while also functioning to maintain an osmotic balance between the internal cell
environment and the external aw.

On a molar basis, glycerol is more effective than other polyols (erythritol, mannitol,
arabitol) for lowering the internal mycelial water potential, hence it is the compatible solute
of choice during extreme low-water stress.
Synthesis of glycerol is regulated via high osmolarity glycerol (HOG)–mitogen-activated
protein kinase (MAPK) stress response pathway.

In Sa. cerevisiae, the principal component of the pathway is the stress-activated MAPK
(Hog1), which is regulated by MAPK kinase (Pbs2).

Changes in osmolarity are sensed by the membrane osmosensor (Sho1), which in turn
signals the activation of Pbs2, resulting in phosphorylation and accumulation of Hog1.

This then triggers the cascade reaction leading to synthesis and accumulation of glycerol.
Salt Loving-Halophiles

Halophiles are organisms that thrive in high salt

concentrations.
They are a type of extremophile organisms.
The name comes from the Greek word for "salt-
loving".
While most halophiles are classified into the
Archaea domain, there are also bacterial halophiles
and some eukaryota, such as the alga Dunaliella
salina or fungus Wallemia ichthyophaga

Because these microorganisms e ectively use hydrocarbons as their sole carbon and
energy sources, they may prove to be valuable bioremediation agents for the
treatment of saline e luents and hypersaline waters contaminated with toxic
compounds that are resistant to degradation.
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Halophiles are capable of growing in salt concentrations of 150 g/L (15 % w/v, 2.5 M).
These organisms are found in all three domains of life, i.e., Archaea, Bacteria, and Eukaryota
(Fig. 1).

Fig. 1 Distribution of halophiles within the three domains of life. Groups annotated in red contain at least
one halophile. The tree was built using small subunit rRNA gene sequences (Ciccarelli et al. 2006).
The hypersaline waters of the Dead Sea was thought to be sterile and lifeless until 1936
following publication of an interesting article in Nature written by Benjamin Elazari titled
‘‘Life in the Dead Sea’’ (Wilkansky 1936).
The renewed interest in hypersaline microbes is, at least partially, attributable to recent
advances in genome sequencing. The first reported genome sequence was for the halophilic
archaeon, Halobacterium NRC-1 (Ng et al. 2000).

Table 1 Classification of microorganisms according to their tolerance to salinity

Halobacterium NRC-1
 The saline/salt content of the Dead Sea makes it Zuni Salt Lake in New Mexico is an example of what is
impossible for most organisms to live there. Only salt know as a Maar. A Maar is a lake formed by some sort of
loving organisms like the halophile can survive in volcanic activity. This lake is a flat saline/salt lake that
environments that have high saline/salt content. formed across the crater of an inactive volcano.

The Great Salt Lake, USA is the saltiest body of water that
is inside a land mass in the western hemisphere.

Adaptations of halophiles to their environments

The survival of microorganisms in hypersaline conditions requires specialized cellular and

enzymatic adaptations to preserve the osmotic balance with the environment.

Cellular adaptation
Microorganisms that are not adapted to highly saline environments will lose water, causing
the cells to first shrivel and subsequently fatal loss of cellular structure as well as function
may occur (Fig. 3a).
To avoid excessive water loss under such conditions, halophiles have evolved two distinct
strategies to increase the osmotic activity of their cytoplasm with the external environment,
1. either producing compatible organic solutes (osmoprotectants; Fig. 3b) or
2. reaching an equilibrium state in which the overall salt concentration within cells
matches that of the environment by accumulating large salt concentrations in their
cytoplasm (Fig. 3c).
Fig. 3 Adaptations of halophiles to hypersaline environments.
(a) The integrity of non-halophile macromolecules is compromised, and the flow of
water out of the cell may cause cell damage.
(b) Moderate halophiles maintain their structures via the synthesis of compatible
organic solutes.
(c) Extreme halophiles maintain their structures via equilibration of cellular and
environmental salt concentrations.
High salt-in strategy

The high-salt-in strategy is an adaptation that protects halophiles from a saline environment
in which they accumulate inorganic ions intracellularly to balance the salt concentration
in their environment.

This process involves Cl- pumps that are found only in halophiles that transport Cl- from
the environment into the cytoplasm. Arginines and/or lysines are positioned at both ends of
the channel to facilitate Cl- uptake and release.

Extreme halophiles of the archaeal Halobacteriaceae family such as H. salinarum,

Haloarcula marismortui, Halococcus morrhuae; and the bacterial Halanaerbiales family
such as Haloanaerobium praevalens, Haloanaerobium acetoethylicum, H. halobius
maintain their osmotic balance by concentrating K+ inside cells.
This is achieved by the concerted action of the membrane-bound proton-pump,
bacteriorhodopsin, ATP synthase, and the Na+/H+ antiporter and results in an electrical
potential that drives the uptake of K+ into cells via a K+-uniport mechanism.

In the absence of light, the Na+/H+ antiporter is replaced by a light-independent Cl-/Na+

symporter, but little is known about the mechanism of such transport.
Low-salt, organic solute-in strategy
The high-salt-in strategy requires physical adaptation of all macromolecules for survival in a highly
saline environment, which is, however, incompatible for the survival of moderate halophiles that
thrive in habitats of fluctuating salinity, i.e., salt concentrations can reach molar levels and then fall
to near-freshwater concentrations after a rainfall.

In this context, a more flexible means to maintain osmotic balance and establish the proper
turgor pressure under different salt environments may be more suitable.

The required adaptations involve evolution of inert compatible organic solutes (osmolytes) in
the halophiles, i.e., solutes that could be derived from the external milieu.

These osmolytes protect the microbial proteins from denaturation in water of low salt concentration
while enhancing the microbes’ tolerance to dramatic fluctuations in the external saline environment.
Glycine betaine in Halorhodospria halochloris was the first reported bacterial osmolyte.
Examples of such solutes and their producing halophiles are listed in Table 3.
Enzyme adaptations
A high-salt environment substantially impacts protein solubility and consequently function.
In such environments, dehydration of cellular proteins becomes pronounced because of
decreased water content in cells owing to water-molecule entrapment within external ionic lattices.
The unfavorable interactions that disrupt internal microbial proteins caused by dehydration may be
averted by modulating their net charge.
A noticeable difference between proteins from halophiles and non-halophiles is that those of
halophiles have a larger proportion of glutamate and aspartate on their surfaces.
Early microbiologists proposed that two primary features: a substantial number of protein charges
and increased hydrophobicity were responsible for the molecular haloadaptation of halophilic
enzymes.
The negativity of the halophile’s proteomes was first revealed by the genome sequencing of
Halobacterium sp. NRC-1 that revealed an acidic proteome (pI. approx. 4.5), unlike that of non-
halophilic microorganisms which proteomes have average pIs’ very close to neutrality.

This hypothesis is supported by the observations that crystal structures of halophilic proteins
include water molecules bound to surface acidic residues.

In addition, their content of hydrophobic amino acids is relatively low.

Such proteins not only are soluble and functional at high salt, they even require molar concentration
of salt for activity and stability
Enzymes of halophiles
Halophilic enzymes are more stable than their non-halophilic counterparts owing to their
polyextremophilic characteristics.
These enzymes remain active in high-salt environments, thermotolerant and alkaliphilic, hence their
popularity for use in biotechnological applications in the food and textile industries and in laundry
detergents.
Examples of halophilic enzymes are:
Amylase a-Amylase, Xylanase, β-Xylanase, β-Xylosidase, Protease CPI, Aldehyde
dehydrogenase, 2-Hydroxy acid dehydrogenase, Endo-1,4-b-Xylanase, Endo-β- Xylanase,
Amylopullulanase, DNase, Alcohol dehydrogenase, Protease, Cellulase, Esterase,
Cyclodextrin glycosyltransferase.
Cell Envelopes of Halophilic Microorganisms
The cytoplasmic membrane of halophilic microorganisms forms the barrier between the cytoplasm
and the environment with its high and possibly fluctuating salinity.
The potential to adjust the membrane according to the external salinity seems to be prerequisite for
halophilic microorganisms.
Alterations of membrane composition have been extensively studied in a range of halophilic
eubacteria.
Many members of the Halobacteriaceae are characterized by the presence of beautiful red orange
pigments, called bacterioruberins, in cytoplasmic membrane, which help to screen out UV radiation
and protect the cells from the harmful effects of sunlight.
Archaeal membrane lipids are unique in containing diphytanylglycerol diether lipids.
Most of the phospholipids and glycolipids of extreme halophiles are anionic so that they impart a
high negative charge density on halophile membranes.