Download IV. Microbial polyhydroxyalkanoates (PHA)

Chap 16 Microbial polysaccharides, single cell oils and
polyhydroxyalkanoates
I. Introduction
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When microorganisms are supplied with excess glucose or other C- or N- sources, they
may produce one or more storage compounds.
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Storage compounds in different organisms:
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yeast and fungi:
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bacteria:
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The storage compounds are usually
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The synthesis of these intra- and extracellular products is promoted when:
II. Microbial polysaccharides
Introduction

Polysaccharides accumulate as storage compounds, e.g. glycogen.


The EPS may remain associated with the cell as a capsule or slime or simply dissolved
in the medium, depending on the chemical structure and how vigorously the culture is
agitated. On solid media, large slimy colonies may be produced.
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
Advantages and disadvantages of microbial EPS:
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Plant polysaccharides are produced on a vast scale and at a low price,
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General Properties:
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Two types:
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Neutral:
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Acidic:
Divalent cations can cross-link polysaccharide chains
to produce a strong gel.
Xanthan (黃原膠)

A large, complex polysaccharide (Mr>106 Da) composed of

Produced from

The most important commercial microbial polysaccharide (20,000 tons/yr). Kelco
(part of Monsanto) is the principal manufacturer.
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Used as
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Widely used for stabilization, suspension, gelling and viscosity control in the food
industry (食品膠).

Can also be used for water-based paints and other domestic and industrial uses.
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Dextran (葡聚糖)
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First commercial microbial EPS by Pharmacia (for 50 yrs)

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Unlike other EPS which are synthesized within the cells,
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The MW of dextran can be manipulated by
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Applications:
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Other EPS: Gellan, Pillulan, alginate…..
Production of polysaccharides

Usually produced in batch culture in stirred tank bioreactors.

Excretion of polysaccharide increases the viscosity of the culture. This limits the
attainable polysaccharide concentration because it becomes difficult to achieve
adequate mixing and O2 transfer. Also, the power requirement is high and the cost of
heat removal to maintain the temperature is increased.

Polysaccharide synthesis generally commences during growth and continues in the
stationary phase.

III.Single cell oils
Introduction
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
Lipids: triacylglycerols composed of three fatty acids attached to a three carbon
(glycerol) backbone.

The cellular roles of lipids include: storage compounds (e.g. fats), components of cell
membrane (e.g. phospholipids), hormones (e.g. steroids).

All lipids share the same common structure but the physical properties vary
enormously, from hard waxy solids at RT (fats) to translucent liquids (oils).

The structure of the fatty acids accounts for the properties of lipids.
Fatty acids: the building blocks of lipids

Nomenclature (Table 16-1):

Trivial names (Palmitic acid: 棕梠酸， -linolenic acid: 亞麻酸 ；arachidonic acid:花生烯酸； DHA：二十二碳六烯酸) are
more commonly used than systematic names (too long).

Numeric designation: e.g. 18:3(n-6). 18: # of carbons in the acyl chain; 3: indicates
the number of double bonds; (n-6) indicates which series the fatty acid belongs to (Fig.
16.18) and denotes the position of the last double bond relative to the terminal methyl
group.

Most commercially important oils (from cells) are produced by eucaryotic
microorganisms such as fungi, yeast as well as algae (prokaryotes tend to accumulate
PHB/A) and normally contain 16-24 carbon atoms and can possess up to 6 double
bonds.
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In general, lipids that are rich in unsaturated fatty acids are liquid at room temperature
whereas lipids containing saturated fatty acids are solids.

When fatty acids are synthesized de novo from acetyl-CoA and malonyl-CoA (also derived
from acetyl-CoA)
by the enzyme complex fatty acid synthase, they are saturated.

Nutritional importance of polyunsaturated fatty acids (PUFAs)



Some diseases (e.g. rheumatoid arthritis, multiple sclerosis, etc.) are associated
with the inability to desaturate fatty acids (though other factors are also involved)
and may be relieved by increased intake of PUFAs.

Two PUFAs, arachidonic acid and DHA, have been implicated in the
development of brain and eye function in new-born infants.
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Microorganism as oil factories

Commercial oils are usually obtained from plants or animals (cheap source). Single
cell oils can only compete with the most expensive specialty oils. Only very long
chain PUFA-rich oils for human consumption for which no convenient plant or animal
source currently exists can fit this purpose.
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Plant sources:
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Can produce essential unsaturated fatty acids,
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
Animal sources:
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Eucaryotic microorganisms source:
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Can produce a variety of PUFAs (see Table 16.3 which lists several organisms for
commercial production)
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Current applications for single cell oils
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Arachidonic acid [20:4(n-6)]
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Processes using this organism has been developed by Zeneca-Roche (UK)
Docosahexaenoic acid (DHA, 22:6(n-3))
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Produced commercially by Martek Biosciences (Maryland, US) using marine
algae C.cohnii, and Omega-Tech (Colorado, in collaboration with Monsanto)
using Schizochytrium. Can be used in baby milk formula.

Fish oil is also a source but it contains 20:5 (n-3) which can not be separated
using conventional oil processing. 20:5 (n-3) can not be used in baby milk
formula.
IV.
Microbial polyhydroxyalkanoates (PHA)
Introduction

PHA are intracellular C- and energy reserve compounds in
many bacteria. They can be isolated from bacteria with
chloroform.

PHA are linear polyesters composed of hydroxyl acid
monomers. 3-hydroxyacid monomers are the most common.


The biodegradability makes PHA of commercial interest for use as package plastics.
PHA as reserve materials in bacteria:
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PHA

Polyhydroxybutyrate (PHB) is the most common among
PHA. PHB is a polyester composed of
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
In batch cultures,
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PHB is also produced when growth of aerobic bacteria is restricted by O2.
PHA composition and properties


Physical properties depend on the constituent monomers. For example,

The composition of PHA is dependent both
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For example,
Regulation of PHB metabolism
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
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Biosynthesis of PHB/V and other PHA

In R. eutropha, PHB/V can be produced from glucose and propionic acid (3HV
monomer is made exclusively from the latter).

For example,
PHA produced from n-octanoic acid or n-octane contains high proportion of 3hydroxyoctanoate.
Biopol-a commercial biodegradable plastic made from PHA

PHB was first found in 1926 by the Institut of Pasteur, but the first patent for
production and recovery was published in the late 1960s.

In the 1980’s, ICI (UK) developed a high-density
fermentation and downstream process for the production
and recovery of Biopol (the trade name used for the
range of polymers manufactured by ICI). The process
was subsequently acquired by Monsanto (US).

Biopol was first used as a shampoo bottle. The greatest
demand was for packaging and disposable products. But
the cost was too expensive to compete with conventional
plastics and production ceased in 1998.

The fermentation of R. eutropha is run in a two-stage
process. The first stage is the biomass production phase
(P is chosen to be depleted at the end because P is
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relatively expensive) and the second stage is the polymer accumulation phase.

Downstream processing involves cell rupture and solubilization of components other
than PHA. The polymer is washed and recovered by centrifugation.
Medical applications:

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The degradation rate of PHB is too low for the plastic to be useful for degradable
sutures (縫線), but can be used for more durable implants, e.g. bone plates, wound
dressings (敷料).

PHB/V and other PHA are not, at present, used in medical applications because the
fate of degradation products is uncertain.
Production of PHA by recombinant bacteria

As long as the genes responsible for the polymer biosynthesis are cloned, E. coli is
suitable for PHA production

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Current research work mostly focuses on the PHB production, but PHB/V and other
PHA have also been produced.
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