FERMENTATION BIOTECHNOLOGY PRACTICAL 4:
BN 304 FERMENTATION
BIOTECHNOLOGY
PRACTICAL 4:
PROPAGATION OF FERMENTATIVE MICROBES: EFFECTS OF OXYGEN AND
CARBOHYDRATE CONCENTRATION
INTRODUCTION
Both bacteria and fungi are capable of carrying out
fermentation metabolism but the fungi are more widely used in fermentation
biotechnology. Owing to the wide use of fungi in fermentation processes, their
energy metabolism has over the years been the subject of intensive research.
Most filamentous fungi of the phyla Basidiomycetes (egMushrooms)
and Zygomycota (eg Rhizopus), Deuteromycetes (eg Penicillium, Aspergillus) are reliant on oxygen as terminal
electron acceptor during their ATP synthesis (oxidative phosphorylation). These
fungi do not grow or grow very slowly in oxygen-poor environments hence can simply
be controlled by keeping oxygen out (nitrogen or carbon dioxide atmosphere).
On the other hand the fungal phylum Chytridiomycota
(eg Neocalimastix, Piromyces) have
members that survive by performing strict fermentative metabolism as they
lack cell machinery (mitochondria) and
enzymes necessary for the TCA cyle and oxidative phosphorylation.
However, members of the phylum Ascomycota (eg
yeasts) are capable of ATP synthesis using either substrate level
phosphorylation (fermentation) or TCA cycle (respiration).
In some cases fungal biomass rather than a
fermentation products is desired as a product.
Examples of this include bakers yeast, Single cell
protein (Pruteen), Quorn Mycoprotein meat substitutes or Saccharomyces
carlsbegnesis for sale to breweries.
Biomasses of filamentous fungi are almost strictly
generated in aerobic submerged cultures whereby propagation is monitored by
protein increases and culture density. Propagation of yeast cultures is on the
other hand carried out using low carbohydrate concentrations (0.5-2%) an
appropriate nitrogen supplement and excess oxygen.
At higher
carbohydrate concentrations, yeasts carry out a fermentation metabolism with
much less ATP production and almost no growth (Crabtree Effect). Interestingly, some yeasts require small amounts
of oxygen to carry out a fermentative metabolism (Kluyver effect).
Industrial baker’s yeast production is therefore
performed in aerobic, sugar-limited fed-batch cultures. The conditions in such
cultures differ drastically from those in the bread environment, which is
anaerobic and with sugars at least initially present in excess.
For the industry carrying out fermentation
processes, a propagation stage for the inoculum is often carried out before the
actual process. This is sometimes termed a Prefermentation and is meant to
achieve
(1) Increase in the number of
cells often a 4-10 fold increase
(2) Adaption of the inoculum to
the fermentation substrate
The monitoring of yeast growth is often done through
microscopy, counting
Chambers at 400magnification.
For the case of fermentative bacteria, the metabolic
complexities of Crabtree and Kluyver are not commonplace hence the optimal
amounts of the propagation carbohydrate are often used. The bacteria inoculum
is then prepared in shake cultures often monitored through absorbance at 660nm
Student
Activity
1. Start by checking out purity
of your own isolates on the microscope at 400x (yeasts) and 1000x (bacteria).
2. Inoculate the Nutrient Broth
provided with your bacteria and take a reading at 660nm as the start. Put on a
shaker at 200 r.p.m and take a reading after every 1 hour plot on a graph
3. For the yeasts make a
suspension through putting one loop-full in the Malt extract Broth provided,
Put a drop on the counting chamber provided, take a reading and submit the
yeast number/ml.
4. Immediately put the
inoculated flasks on the shaker at shake for 16 hours over-night at 150-170rpm.
Again use the counting chamber to note the reading and submit.
5. Keep the propagated Biomass
in the fridge for fermentation experiments next week.
