Handbook of Functional Lipids

from major culture collections; no attempt was made to isolate possible organisms
from the environment.

The criteria that were used to evaluate performance in this screening process

were as follows:

•

The organism should grow readily in submerged culture attaining at least
10 g/l in 3 d or less in simple stirred vessels. It should not pose problems
for extensive filamentous growth or for pellet formation.

•

It should have an extractable oil content of not less than 20% of the
biomass.

•

It should preferably have a GLA content of the total fatty acids of as near
to 20% as possible and, preferably, over this value.

•

The oil should be over 90% triacylglycerol.

•

The organism should pose no hazard for large-scale cultivation. It should
not be an animal pathogen or a plant pathogen. It also should have no
history of causing any allergic reaction or toxicity.

•

Ideally, the organism should grow at 30

C or slightly higher because if it

had a lower growth temperature requirement, this could otherwise incur
additional expenditure for cooling large-scale cultures.

After an initial cultivation in shake-flask cultures, all organisms were grown in
vortex-aerated 1 l bottles [22]. In all cases, a glucose-based medium was used and,
as the pH was not controlled in these vessels, diammonium tartrate was used as the
N source

with small amounts of yeast extract, malt extract, and peptone (totaling

0.5 g/l) added as sources of any vitamin that may be required. (Ammonium tartrate
was used as the N source because this prevents acidification of the medium that
occurs when ammonium chloride or ammonium sulfate is used, as the uptake of the
ammonium ion leaves HCl or H

behind that quickly acidifies the medium and

prevents microbial growth.) The medium composition was carefully balanced so that
it would become N-limited after about 24 to 30 h (see Figure 2.1); a C:N ratio of
40:1 was therefore selected as able to induce lipid accumulation in an oleaginous
microorganism.

After evaluating the performance of over 200 organisms, it seemed that the most

promising species lay in the genera of

Cunninghamella

and

Mucor

, with

Rhizopus

stolonifer

a further candidate. Accordingly, extensive examination then began of

the key species in each of the two likely genera by looking at the performance of
as many strains of each species as could be obtained from culture collections
worldwide. Table 2.2 lists the results of 57 individual strains taken from just 8
species.

As can be seen, the lipid and GLA content of the fatty acids could vary enor-

mously within a single species: for example, with

Mucor

circinelloides

the content

of lipid in the cells, all grown under identical conditions, ranged from 3 to 37% and
the GLA ranged from 8 to 32%. This wide divergency indicates that screening can
often miss the best organism, and therefore one should be prepared to examine the
complete range of strains that are available in order to ensure that the best organism
is not overlooked.

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