MKBS 314

NKANYEZENHLE THANDEKA NDLOVU

32282877
 Question 1

i. Molecular method- a PCR based method: Denaturing and temperature gradient gel
electrophoresis (DGGE and TGGE)
ii. DNA is extracted from the environmental sample and purified.
DNA extraction was carried out using the modified Holben’s method.

1. Duplicate soil samples of 5g each (pre-washed with 0.1M sodium phosphate) were mixed
by vortexing them in the presence of large and small beads (SIGMA washed) 0.15g sodium
dodecyl sulfate (SDS), and 15 ml of 1 mM sodium phosphate (pH 8.0). They were then
incubated for 1 h in a 65 ºC water bath with end-over-end inversions every 10 min, shaken for
30 min at a speed of 250 cycles per min, and centrifuged at 8000g for 15 min. SDS was
precipitated by centrifugation at 8000g for 10 minutes at 10 C after the supernatants were
incubated on ice for 30 minutes. The cleared lysate (10 ml) was extracted with an equal
volume of phenol-chloroform–isoamyl alcohol (25:24:1) and then with chloroform–isoamyl
alcohol (24:1). The aqueous phase was recovered by centrifugation and precipitated with 0.6
volumes of cold isopropanol after freezing at 80 C for 1 h. The pellet of crude DNA was
obtained by centrifugation at 16,000g for 20 min at 2 C, washed with cold 70% ethanol, and
suspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The crude DNA was
incubated for 15 min with 1/5 volume of 8 M potassium acetate on ice followed by
centrifugation at 13,800g, 4 C for 20 min. The supernatants were re-extracted with phenol-
chloroform–isoamyl alcohol precipitated with isopropanol and resuspended in EB buffer (10
mM Tris–HCl, pH 8.5).

Amplification
18S rDNA PCR amplification was carried out using the fungal-specific primers, EF4f (50 -
GGA AGG G [G/A] T GTA TTT ATT AG-30) and Fung5r (50 -GTA AAA GTC CTG GTT
CCC-30 ) designed by Smit et al. [11] and commercially synthesized (Sigma Genosys,
Australia). The reaction mixtures were 50 ll which usually contained 1· PCR buffer, 5 mM
MgCl2, 2 mM dNTPs, 2.5 U Taq polymerase from GibcoBRL, 10 lM each of primers, and 1
ll (10 ng) DNA template. The thermal cycling scheme was heated to 94 C for 3 min; then 35
cycles were run at 94 C for 1 min, 55 C for 1 min, 72 C for 2 min; and finally, 72 C for 10
min.
TGGE Screening
TGGE screening of the PCR products (550 bp fragments) was conducted at 300 V with a
temperature range of 27–54 C on a QIAGEN TGGE-System (QIAGEN Gmbh, Hilden,
Germany). The polyacrylamide gel was composed of 5% (wt/vol) acrylamide, 0.17% (wt/vol)
 bisacrylamide, 8 M urea, 2% (wt/vol) glycerol, 1· ME buffer, 20 mM 3-[N-morpholino]
propane sulfonic acid (MOPS), 75 ll tetramethyl ethylenediamine (TEMED) and 136 ll of
10% (wt/vol) ammonium persulfate (APS) according to the manufacturer’s instruction. The
gel was cast onto a gel support film (Gelbond PAG, BMA, Rockland, MD) and polymerized
for 60 min. 8 ll samples including 2 ll template DNA, 4 ll of 8 M urea, 0.8 ll of 10· ME + dye
buffer, and 1.2 ll H2O were loaded into the gel and run in 1· I buffer for 4 h. Gels were then
silver-stained according to the manufacturer’s protocol. SSCP screening of the PCR products
was conducted at 300 V on a QIAGEN TGGE-System. One microlitre of the PCR product
was mixed with 4 ll formamide loading dye (80% formamide, 10 mM EDTA, 0.1% xylene
cyanol FF, and 0.1% bromophenol blue, pH 8.0), denatured at 95ºC for 5 min and
immediately chilled on wet ice, then the previous denaturation step was repeated [19]. The
denatured samples (8 ll) were then loaded into the slots of the polyacrylamide gel and run for
14 h at room temperature, and finally, the gel was silver-stained. The polyacrylamide gel was
composed of 8% (wt/vol) acrylamide, 0.21% (wt/vol) bisacrylamide, 5% (wt/vol) glycerol,
0.5 · TBE buffer (45 mM Tris–borate, 1 mM EDTA, pH 8.0), 93 ll TEMED and 232 ll 10%
(wt/vol) APS, with a total volume of about 46.5 ml. The gel was cast onto a gel support film
(Gelbond PAG, BMA, Rockland, MD) and polymerized at least for 60 min.

iii. A simple way to facilitate the identification of a particular micro-organism is to provide it with
one or more genetic markers that are unlikely to be widely distributed in the natural environment
(Pickup, 1991). The molecular techniques generally involve the extraction of nucleic acid,
directly or indirectly, from the soil (Nannipieri et al., 2003). It is further explained by Nannipieri
et al. it is independent of culture, and according to their sensitivity can detect species, genera,
families, or even higher taxonomic groups. DGGE is rapid, reliable, and reproducible. Many
samples can be analysed simultaneously.

i. Enumeration by plate count technique.

ii. This methodology is provided in the Manual for soil analysis-monitoring and assessing soil
bioremediation (2005).
1. Pipette 0.1mL or 1mL of inoculum onto three or five replicate agar plates and spread it
uniformly with a sterile glass rod. Once the inoculum is absorbed, place bags in a plastic bag
to maintain high humidity.
2. Incubate at a suitable temperature and atmosphere and observe the appearance of colonies
on plates with 30-300 colonies (Koch 1994).
 3. Count the colonies on replicate plates with 30–300 colonies, compute the mean, and
calculate the CFU per gram dry mass in the original sample using the dilution factor and dry-
mass correction factor.
4. To enumerate colonies that can grow on liquid hydrocarbons spread a small volume (e.g.,
50 µL per plate) onto the surface of mineral medium agar either before or after inoculation,
leaving small droplets on the agar surface.
iii. The plate count technique quantifies the viable microbes in a sample by counting the number
of colonies that form on or in a solid growth medium inoculated with dilutions of that sample.
Each colony is assumed to have originated from a single propagule or “colony-forming unit”
(CFU), whether that be a bacterial cell, endospore, hyphal fragment, or spore. A non-selective
growth medium may be used to cultivate generalists, or a selective medium may be used to
enumerate specialists such as hydrocarbon-degrading bacteria and fungi. Davis et al. (2005)
suggest that plates with a minimum of ten colonies per plate rather than 30–300 colonies
should be used for enumeration. This reduces depression in viable counts due to over-
crowding of colonies on plates leading to inhibition of some species by others, or the
depletion of nutrients by fast-growing colonies that prevents slow growers from reaching a
countable size.

i. Enumerated by microscopy
ii. This methodology is provided in the Manual for soil analysis-monitoring and assessing soil
bioremediation (2005).
Using a sterile spatula, place 10 g of soil into the first dilution vial holding 90 mL of diluent
and record the precise wet mass of the sample put on a top-loading balance. This is the 101
dilutions. To limit the impacts of sample heterogeneity, it was recommended by Alef and
Nannipieri (1995) to use 20 g of soil in 180 mL of diluent. To express the counts in terms of
soil dry mass, pour a similar sample into a tared balance weigh, and repeat three times to
ensure accuracy. Overnight, dry the sample at 105ºC to a constant mass and record the mass.
Shake or mix the dilution container aggressively it can be manually or mechanically to disrupt
soil aggregates; recommended periods range from 1 minute to 1 hour and can be empirically
optimized for soils. Dilute tenfold by moving a 10mL sample from the dilution container's
center to a new 90mL dilution bottle, or a hundredfold by transferring 1.0 mL into 99 mL of
diluent. Mixing between dilutions can be done by hand by shaking the bottle transfers, or with
a vortex mixer. Continue with ten-fold serial dilutions suitable to the enumeration method,
 e.g., for aerobic heterotrophs in uncontaminated agricultural soils dilute to 109 for the most
probable number and 107 for plate counts.
iii. To reduce the bias inherent in culture-based enumeration methods, total counts of microbes in
the soil can be observed directly using microscopy (Fry, 1990). Direct enumeration methods
can provide the total number of cells (live plus dead) or may discriminate between live and
dead cells (Foght & Aislabie, 2005).

Question 2

Malolactic fermentation is important in the vinification process. This process, also called biological
deacidification, consists of the decarboxylation of L (−) malic acid to L (+) lactic acid as well as CO2
(Lasik, 2013). MLF in wine is done for three reasons. First, the wine is deacidified, resulting in a rise
in pH; second, malic acid is removed as a possible carbon substrate, contributing to microbial
stability; and third, the wine scent profile is altered (Davis et al., 1988; Kunkee, 1991, Ugliano et al.,
2003). During MLF, bacteria alter the final fragrance balance by changing fruity scents and perhaps
creating aroma active chemicals (Henick-Kling, 1995).

This fermentation improves the biological stability of wines by preventing the use of malic acid by
undesirable species (Davis et al., 1985). Another beneficial property of malolactic bacteria is the
breakdown of unwanted by-products of alcoholic fermentation, such as acetaldehyde. Lasik (2013)
explains that they considerably reduce the concentration of this poisonous, easily volatile molecule,
which is highly undesirable in terms of sensory and health. Apart from its poisonous qualities,
acetaldehyde has a high SO2 binding capability, making it an antibacterial and antioxidative
molecule. Conversion of SO2 from free to bound form (e.g., with acetaldehyde) is a serious issue in
winemaking methods requiring sulfitation process intensification. Thus, MLF treatment may help to
reduce wine sulfitation and effectively reduce acetaldehyde levels (Henick-Kling, 1993). Increments
in total higher alcohols, ethyl esters, and acids enhance the sensory properties and quality of wines
that have undergone MLF. Wines that had undergone the MLF showed a significant increment in total
higher alcohols, esters, and acids that are important in wine's sensory properties and quality. This is
the reason to suggest the induction of MLF in red wines with selected lactic acid bacteria strains that
can offer a positive contribution to the final aroma in wines (Maicas,1999).
References

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