123 5. THE PROMOTION OF SALT QUALITY THROUGH ALGAL INOCULATION IN PUTHALAM SALTWORKS 5.1. INTRODUCTION The salt was the extremely precious thing in the ancient times and it was the wealth symbol called as “The white gold”. Solar saltworks are most efficient converters of solar energy into an inorganic commodity. Salt is mainly a basic inorganic chemical raw material. In the field of inorganic chemistry solar salt production is truly a remarkable and uniquely efficient process (Sedivy, 2009). Designing the saltworks is the most important aspect. In old saltworks environmental aspects were not taken into consideration. Modernization of saltwork will help us to have a healthy and stable ecosystem (Moosvi, 2006). The concept of the saltern ecosystem and the relationship between the saltern ecosystems and salt production was first proposed by Carpelain in 1950s. It has been proved that the biological management and process control of solar salt production at all levels of the production area ensure the balance of the concentrations of volume of brine and maintain the balance of the entire saltern ecosystems. In this way the maximum benefit from solar salt production fields in terms of salt quantity and quality can be reached (Kurian et al., 1988; He et al., 2009). The mode of operation of solar salt production plants is very similar worldwide and the composition of seawater from which the salt produced is nearly identical, the size and quality of the salt crystals formed in the crystallizer ponds of salt production facilities around the world is highly variable (Oren, 2009).
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5. THE PROMOTION OF SALT QUALITY THROUGH
ALGAL INOCULATION IN PUTHALAM SALTWORKS 5.1. INTRODUCTION
The salt was the extremely precious thing in the ancient times and it was the
wealth symbol called as “The white gold”. Solar saltworks are most efficient
converters of solar energy into an inorganic commodity. Salt is mainly a basic
inorganic chemical raw material. In the field of inorganic chemistry solar salt
production is truly a remarkable and uniquely efficient process (Sedivy, 2009).
Designing the saltworks is the most important aspect. In old saltworks
environmental aspects were not taken into consideration. Modernization of
saltwork will help us to have a healthy and stable ecosystem (Moosvi, 2006).
The concept of the saltern ecosystem and the relationship between the
saltern ecosystems and salt production was first proposed by Carpelain in 1950s. It
has been proved that the biological management and process control of solar salt
production at all levels of the production area ensure the balance of the
concentrations of volume of brine and maintain the balance of the entire saltern
ecosystems. In this way the maximum benefit from solar salt production fields in
terms of salt quantity and quality can be reached (Kurian et al., 1988; He et al.,
2009). The mode of operation of solar salt production plants is very similar
worldwide and the composition of seawater from which the salt produced is nearly
identical, the size and quality of the salt crystals formed in the crystallizer ponds of
salt production facilities around the world is highly variable (Oren, 2009).
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Microalgae can be large-scale cultivated either by open or close culture
system or closed system (Ugwu et al., 2008). Salt deposits accumulate on every
continent and are distributed in two great belts, one in either hemisphere, where lie
approximately between the latitudes of 15º and 35º from the equator. Today,
sodium chloride (Halite) is a cheaply produced commodity extracted either from
mines or saltpans. Salt crusts first precipitated on the floor as upward growing
cubic crystals (Al-Juboury et al., 2009). Most of the world’s salt supplies are
obtained by solar evaporation of seawater which contains on average 3.6%
dissolved salts, of which sodium chloride comprises 77%. Salt is harvested by
exposing seawater to the action of sun and wind in a chain of concentrating ponds
to the point where it becomes saturated by evaporation with common salt. The less
soluble salts iron oxide and calcite, followed by gypsum are precipitated out at this
stage (Hough, 2008). Solar evaporation is the oldest method of salt production
which depends on certain factors. They are: 1) large area with small slope, 2) low
salt permeability, 3) low rainfall rate, 4) high evaporation, 5) dry wind and 6) long
summer season for evaporation (Calvinaco, 1990).
A saltwork is defined as a flat and location where facilities have been
constructed to control seawater or brine inlet and arranged a brine flow through
evaporating ponds, in accordance with calculated parameters such as brine flow
rate, rate of evaporation, specific gravity of brine, with the view of concentrating
the seawater sequently by solar evaporation until salt is crystallized in crystallizing
pans (Garcia, 1993). Brine concentration promoted by evaporation of its water
content, results on the successive precipitation of the least soluble salts, CaCO3,
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CaSO4 followed by the production of sodium chloride and finally magnesium and
potassium salts (Collares-Pereira et al., 2003).
In many saltworks, hydrobiological activities and their management are
essential for the production of high quality and quantity salt. One of there is the
naturally available organic and inorganic nutrients support the development of
Dunaliella salina algal blooms which are beneficial for solar salt heat absorption
resulting in rapid evaporation. This leads faster production of high quantities of salt
crystals (Rahaman and Jeyalakshmi, 2009a) and an increase in the salt quality
(Reginald and Diana, 2008). Prokaryotes may survive inside fluid inclusions for
tens of thousands of years using carbon and other metabolites supplied by the
trapped microbial community, most notably the single-celled alga Dunaliella, an
important primary producer in hypersaline systems (Lowenstein et al., 2011).
Dunaliella a wall-less unicellular biflagellate, naked green alga (Chlorophyta,
Chlorophyceae) is a dominant photosynthetic organism in many extreme conditions
and it has high tolerance under altering environmental factors (Jimenez and Niell,
1990). The genus was first described by Teodoresco (1905) with the type species
being Dunaliella salina, and is named in honour of M.F. Dunal, who was the first
to recognize that the red colour of certain hypersaline reservoirs was caused by an
alga (Dunal, 1837). Dunaliella salina reproduce fastest at intermediate salinities,
continues to divide slowly after they flow downstream to the high salinity ponds
and crystallizers. The algae develop massive populations, release significant
quantities of organic substances (Giordano et al., 1994) and colour the brine
yellow-orange to bright orange (Davis, 2009). The population density of
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Dunaliella in crystallizer ponds varies greatly according to geographic location,
nutrient status and management the salterns. Dunaliella uses sunshine,
carbondioxide, waste or brine water with natural nutrients. It can use green energy
for supporting biomass growth such as solar and wind (Regunathan and Kitto,
2009). The cell shape of the genus Dunaliella is very variable being oval, spherical,
cylindrical, ellipsoidal, egg-pear or spindle shaped with radial, bilateral or
dorsiventral symmetry or being asymmetrical. Dunaliella salina appears red when
cultured at high salinities. Because of their ability to grow and time at high
salinities Dunaliella species are the predominant algae in saltworks and naturally
occurring hypersaline environments. However, too extensive development of the
biota may lead to the production of poor quality salt (Davis, 1979; Davis and
Giordano, 1996). Kanyakumari District of Tamil Nadu contributes a little to the
overall salt production of the state. The saltworks are of a few hectares in area,
owned by different licensees. The salt workers follow their own method of
manufacture, mostly traditional (Rose, 2007). The aim of this research was to
study the influence of Dunaliella salina cells in the production of maximum pure
salt in Puthalam saltworks for two years.
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5.2. MATERIALS AND METHODS
5.2.1. Sampling and microalgae isolation
To understand the role of Dunaliella salina in quality salt production the
brine samples were collected from the condenser pond of Puthalam saltworks. The
bulk availability of Dunaliella salina and its rich growth in Puthalam saltworks
made to select a study on inoculation. For the isolation of microalgae Dunaliella
salina, the isolates of bacteria from salt pans after identification were inoculated in
the laboratory. The collected samples were enriched with Walne’s medium
(Walne, 1974) and allowed the samples to grow for three days in the exposition of
100 lux light and proper sterilized aeration. After three days, 1 ml of sample was
pipetted out from the culture and serially diluted upto 10–8 using sterilized
seawater.
5.2.2. Algal culture and growth conditions
For stock culture the microalgae Dunaliella salina were sub cultured in test
tubes containing Walne’s medium which prepared with sterilized seawater. The
culture period was 7 days with continuous illumination provided by lamps, till
getting in a good population of algal cells (Plate 5b).
The test tubes which showed a good growth of Dunaliella salina cells
(Plate 5c) were transferred to 200 ml conical flask containing 150 ml of culture
media with triplicate. Then the inoculation was multiplied by frequent inoculation
in large volume of 2 litre jars. After getting 2 litre of culture inoculums, they were
transferred to the transparent 5 litre pearl pet jars. Proper aeration was given for the
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algal growth. It is important for microalgae culture because 1) air is a source of
carbon (from CO2) for photosynthesis, 2) CO2 provides essential pH stabilization
and 3) physically mixing the culture, keep nutrients and cells evenly distributed,
reduces self-shading and/or photoinhibition. Air diffusers (air stones) create small
bubbles that maximize oxygen/CO2 transfer and they are often used for small
volume cultures. The culture was maintained at a constant temperature of 27 ± 1°C
under illuminated with 6 flourescent lamps and salinity of 100‰. The pH was
measured every day by Hi-Indicator pH paper (2 to 10.5). The optimum growth
temperature for Dunaliella salina is in the range of 20 to 40°C (Borowitzka, L.J.,
1981) depending on the strain. There is also a strong relation between the growth
rate, temperature and salinity (Gimmler et al., 1978) and between light intensity
and temperature tolerance (Federov et al., 1968). The optimum pH was ranged
from 7 to 9. After getting good algal growth, the Dunaliella salina culture was
made ready for inoculation into field experimental pond selected in the study area.
Eight condenser of same size and crystallizer ponds (15 x 12 m) were
selected for the practical application in the field study. Among them four ponds
were considered as control and the other four as experimental ponds. Dunaliella
salina algal stock inoculation was set only in experimental ponds.
5.2.3. From the laboratory to the field
The microalgae inoculums developed in the laboratory (Plate 5a) were
brought to the field (saltpan) for inoculation. The inoculation was carried out
between 6 and 6.30 a.m. The microalgae in 1.5 litre culture were inoculated
randomly in different places of the each experimental units. Water samples were
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collected in different places of the experimental and control ponds to estimate the
microalgae biomass soon after inoculation. From the day of inoculation onwards
the water samples were regularly collected and brought to the laboratory. This
process was continued till the brine water attained 100 ppt salinity in condenser.
This was reached on the 7th day after inoculation in all the seasons of the two years
study period. The algal count in control and experimental inoculation
quantified for 7 days with haemocytometer using light microscope. It was
calculated for all the seasons throughout the study period and they were tabulated
in Table 5.1 and 5.2.
When the water salinity reached 100 ppt, the brine water was allowed to
four experimental crystallizer ponds separately (Plate 5f and 5g). Likewise the
brine water in the control ponds was also allowed to another four crystallizer
ponds, where the brine water was allowed for crystallization. After the
crystallization process (salt formation) was over, salt samples (crystals) from both
control and experimental ponds were carefully collected in separate polythene bags
(Plate 5h and 5i). The salt samples so taken were immediately transferred to a dry
cleaned airtight container covered with a polythene bag and they were brought to
the laboratory for the salt quality analysis. The same experiment was conducted for
all the four seasons of the two years study. In Puthalam saltworks, the crystals
were heaped up in pans. The salt was harvested and dumped into concrete floors
alongside the salt pan to dry out in the sun and it was transferred to the packing
area where it is packed by hand. The package are loaded into trucks and
transported out (Plate 5m). Standardized salt is protected by covering it with
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tarpaulin during rainy season. Collection, storage and transport of salt is a highly
labour-intensive task in saltworks.
5.2.4. Salt quality analysis
5.2.4.1. Determination of moisture content
Fifty grams of salt sample was taken and powdered well using a mortar and
pestle. About 20 g of powdered salt was weighed and transferred into a bottle
which was previously dried and weighed. The sample was kept in a hot air oven at
140ºC to 150ºC for four hours. Then it was cooled in desiccators and weighed to
constant weight. The percentage of moisture content was calculated by using the
formula given below:
M2 – M1 % of moisture content = X 100 M1
Where M1 is the initial mass of the sample and M2 is the final mass of the sample
taken for test.
5.2.4.2. Determination of insoluble matter
Ten grams of the dried salt sample was weighed and dissolved in 100 ml of
distilled water in a beaker and heated to boiling. Then it was allowed to cool and
filtered through Whatman No.1 filter paper. The residue was washed till it was free
from soluble salts. Then the filtrate was collected, washed and made up to the
mark in a 500 ml standard measuring flask with distilled water. The solution was
preserved for subsequent analysis. The crucible/filter paper was dried along with
the insoluble residue to constant weight.
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M3 x 100 % of insoluble matter = M1
Where M1 is the initial mass of salt sample taken for test and M3 is the mass of the
insoluble matter.
5.2.4.3. Preparation of 0.1N sodium chloride stock solution
Ten grams of sodium chloride (analar) was weighed in a watch glass and
heated in an air over at 100ºC to 110ºC for about an hour, subsequently cooled in a
desiccator. From this sample taken 1.5 g in a clean, dried, previously weighed
bottle and determined its correct weight (mass). Then the salt sample was
completely transferred into a 250 ml volumetric flask and made up the solution to
the mark. This was the standard stock solution. Sodium chloride was estimated by
M x 4 = 58.46
Where, M is the mass of sodium chloride taken, 58.46 is the equivalent weight of
NaCl, 4 is the factor to convert the NaCl content per litre of the solution.
The comparison of sulphate, calcium and magnesium of the salt samples
were estimated using the stock solution as per the standard procedures explained in
the third chapter.
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5.3. RESULTS
5.3.1. Dunaliella algal count in the experimental and control ponds
during the study period (from March 2009 to February 2011)
The algal population (Dunaliella salina) in the control (uninoculated) and
experimental (inoculated) ponds were estimated by using Hemocytometer and the
results during the study period are presented in the Figure 5.1. and 5.2. It was
carried out for seven days from the date of inoculation for both years. The first
reading was calculated on the day of inoculation (0 day). On this day Dunaliella
salina algal count was 0.16 ± 0.001 x 104 cells/ml. The other estimations for the
subsequent seven days were 0.40 ± 0.012, 0.96 ± 0.021, 1.32 ± 0.046, 2.40 ± 0.002,
3.98 ± 0.015, 4.51 ± 0.024 and 5.02 ± 0.013 x 104 cells/ml from the first to seventh
day respectively. But there were no algal count for two days from the day of
inoculation in control ponds. And it was 0.22 ± 0.001, 0.97 ± 0.005, 1.35 ± 0.012,
1.89 ± 0.064 and 2.42 ± 0.321 x 104 cells/ml respectively from 3rd to 7th day for the
first year study on season I.
In the second year study also, the algal count was estimated as same as last
year. The experimental pond showed the algal count (Dunaliella salina) was 0.10