Selection of Tolerant Heavy Metal Yeasts from Different Polluted Sites.

Introduction.
As a result of many industrial and waste-disposal operations, our environment is
becoming more and more contaminated by heavy metals—mercury, chromium,
arsenic, cadmium, nickel, copper, and so on.
Water and soil pollution has become a major concern in the world, as much of the
population relies on groundwater for drinking and irrigation. Heavy-metals
contamination can cause metal toxicity in animals and humans.
Trace elements such as cadmium, copper, mercury, or arsenic are very toxic heavy
metals and have been found in the human environment at increased concentrations
because a wide variety of industrial activities have accelerated the release of these
metals at higher rates than natural geochemical cycling processes can tolerate.
Although many heavy-metal cations are essential components of biological
systems, all are potentially toxic. They play an important role in sophisticated
biochemical reactions such as nitrogen fixation, water cleavage during oxygenic
photosynthesis, respiration with oxygen or nitrate, hydrogen assimilation, cleavage
of urea, transcription of genes into mRNA, and so on. These reactions are based on
catalysis by biochemical heavy-metal complex compounds. Essential or not, most
heavy-metal ions are toxic at higher concentrations.
The environmental mercury cycle is mediated by both geological and biological
processes. Mercury compounds are leached from natural sources by weathering
rocks and soil. However, the sources of environmental contamination of mercury
arise from human activities, such as burning coal and petroleum products, the use
of mercurial fungicides in papermaking.
Objective:
The objective of this laboratory protocol is to describe procedures used for yeast
isolation from several sites contaminated with industrial flow out containing heavy
metals, with the main purpose of isolating yeasts able to live in these unfavorable
environments.
Materials
Materials, Habitats
1. Material for isolating mercury-tolerant yeasts: agricultural and papermaking soil
contaminated with petroleum products and mercurial fungicides.
2. Material for isolating chromium-tolerant yeasts: wastes from tanneries and dye
factories.
3. Material for isolating arsenic-tolerant yeasts: soil contaminated with pesticides
and sediment from an arsenic-endemic area.
4. Material for isolating cadmium-tolerant yeasts: soil and water contaminated with
spent nickel-cadmium batteries.
5. Material for isolating copper-, mercury-, cadmium-, chromium-, iron-, nickel-,
and lead-tolerant yeasts: soil and sediments exposed to effluents from mining and
other industries.
Culture Media and Solutions
1. Enrichment solution: 1 g/L glucose and 0.5 g/L yeast extract.
2. Isolation medium: 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L
dextrose, 20 g/L agar (YM, Difco); plus 100 mg/mL of tetracycline, erythromycin,
and chloramphenicol.
3. Maintenance culture medium: 10 g/L yeast extract, 20 g/L peptone, 15 g/L
dextrose, and 20 g/L agar (YEPD).
4. Base culture medium for determining heavy-metal tolerance: 6.7 g/L yeast
nitrogen base (YNB, Difco), 10 g/L dextrose, and 1.5 g/L agar. It is used as a
minimal medium.
5. Heavy-metal stock solutions: 1 M CuSO4, NiSO4, CdCl2, K2Cr2O7, As2O3,
As2O5, and HgCl2