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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

± 0.003, 0.26 ± 0.010, 0.97 ± 0.055, 1.64 ± 0.021, 2.39 ± 0.183, 3.10 ± 0.019, 3.95

± 0.036 and 4.73 ± 0.054 x 104 cells/ml from 0 to 7th day respectively. At the same

time no algal count was reported in control pond from 0 to 2nd day. And algal

count noticed from 3rd to 7th day was 0.30 ± 0.012, 0.56 ± 0.023, 1.14 ± 0.065, 1.89

± 0.009 and 2.66 ± 0.109 x 104 cells/ml.

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In season II the algal count showed the results of 0.09 ± 0.003, 0.36 ±

0.015, 0.97 ± 0.032, 1.51 ± 0.067, 2.00 ± 0.148, 2.87 ± 0.095, 3.92 ± 0.227 and

4.66 ± 0.301 x 104 cells/ml from the day of inoculation to 7th day respectively. But

in the control pond, there was no algal count from 0 to 2nd day. The algal count in

the subsequent days were 0.04 ± 0.003, 0.75 ± 0.071, 1.12 ± 0.063, 1.96 ± 0.038

and 2.58 ± 0.076 x 104 cells/ml during the first year study.

During season II of the second year study, the algal density in the

experimental pond was 0.07 ± 0.002 x 104 cells/ml on the day of inoculation. The

other estimations for the subsequent days were 0.65 ± 0.004, 1.43 ± 0.015, 2.07 ±

0.069, 2.90 ± 0.124, 3.66 ± 0.052, 4.25 ± 0.236 and 5.13 ± 0.405 x 104 cells/ml

respectively. But no algal count was observed in control pond up to 3rd day. After

that the algal count was 0.05, 0.82, 1.67, 2.30 and 2.91 x 104 cells/ml for the other

days.

Dunaliella salina population in the inoculated and uninoculated (control)

pond during season III of the first year were calculated. On the first day the algal

count of the inoculated (experimental) pond was 0.05 ± 0.006 x 104 cells/ml. The

other counts on seven days were 0.62 ± 0.002, 1.22 ± 0.045, 1.76 ± 0.032, 2.19 ±

0.106, 2.96 ± 0.078, 3.54 ± 0.456 and 4.09 ± 0.319 x 104 cells/ml. But the algal

count in control pond was 0, 0, 0, 0.02 ± 0.000, 0.53 ± 0.006, 1.27 ± 0.095, 1.95 ±

0.394 and 2.24 ± 0.085 x 104 cells/ml respectively for the following days.

Season III of the second year study showed the algal count of the

inoculation day was 0.16 ± 0.005 x 104 cells/ml in the inoculated ponds. Also the

other estimations on the succeeding days were 0.48 ± 0.002, 1.05 ± 0.011, 1.87 ±

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0.008, 2.16 ± 0.176, 2.77 ± 0.095, 3.65 ± 0.210 and 4.43 ± 0.153 x 104 cells/ml. But

algal count in control pond was 0, 0, 0, 0.12 ± 0.003, 0.98 ± 0.017, 1.83 ± 0.073,

2.20 ± 0.118 and 2.92 ± 0.036 x 104 cells/ml.

During season IV of the first year study, the algal count of the experimental

pond showed the reading of 0.08 ± 0.011, 0.21 ± 0.003, 0.95 ± 0.062, 1.49 ± 0.078,

2.03 ± 0.142, 2.88 ± 0.027, 3.66 ± 0.128 and 4.01 ± 0.035 x 104 cells/ml

respectively from 0 to 7th day. But the algal count in the control pond was 0, 0, 0,

0.03 ± 0.001, 0.72 ± 0.025, 1.38 ± 0.072, 2.05 ± 0.160 and 2.3 ± 0.237 x 104

cells/ml respectively from 0 to 7th day of the estimation.

Dunaliella salina algal counting on the control and experimental pond were

estimated during season IV of the second year was studied. On the day of

inoculation, the algal count was 0.08 ± 0.003 x 104 cells/ml. The other estimations

for the seven days were 0.14 ± 0.006, 0.96 ± 0.000, 1.54 ± 0.001, 2.26 ± 0.004,

3.05 ± 0.096, 3.84 ± 0.185 and 4.22 ± 0.075 x 104 cells/ml from 0 to 7th day. But

the algal count in control pond was 0, 0, 0, 0.03 ± 0.000, 0.75 ± 0.008, 1.26 ±

0.046, 2.11 ± 0.065 and 3.80 ± 0.304 x 104 cells/ml respectively for the following

days..

5.3.2. Salt quality parameters during the first year study period

The characteristics of salt samples obtained from the control and

experimental salt ponds were showed in Table 5.3 and 5.4. The samples of salt

crystals harvested from both experimental and control crystallizer ponds were

analyzed separately for the chemical nature such as moisture content, insoluble

matter, sulphate, calcium, magnesium and sodium chloride for different seasons in

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the study period (Fig. 5.3 to 5.14). The day of salt is harvested often depends on

the size of salt crystals and also the experience of the operator. The salt quality

parameters of the salt samples were analyzed during the study period (March 2009

to February 2011) are described below.

5.3.2.1. Moisture content (%)

Data on the moisture content of the salt samples showed the decreased level

from the control towards the experimental ponds in all the four seasons of the first

year study. In season I, the moisture content in the salt samples of the control pond

was 6.06 ± 0.013% and the salt from experimental pond was 5.92 ± 0.015%. The

statistical analysis (‘t’ test) made on the presence of moisture content between the

salt samples in the control and experimental ponds were statistically significant

(t = 7.72315; P < 0.05). In season II, the moisture content of the salt sample was

5.20 ± 0.033% in control pond and 3.06 ± 0.055% in the experimental pond. The

‘t’ test made for moisture content between the salt samples from the control and

experimental ponds were statistically significant (t = 60.02164; P < 0.05).

During season III, the moisture content was 6.18 ± 0.049 and 5.20 ± 0.082%

in control and experimental ponds respectively. The ‘t’ test made on the presence

of moisture content between control and experimental salt samples were

statistically significant (t = 56.4460; P < 0.05). Similarly during season IV, the

moisture content of control salt sample was 3.82 ± 0.088% at the same time in

experimental pond it was 3.66 ± 0.149%. The statistical analysis (‘t’ test) showed

the moisture content between the salt samples of control and experimental pond

were statistically non-significant (t = 1.72758; P < 0.05).

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The next year of the study period, the moisture content in the salt samples

showed the decreased level from the control towards the experimental ponds except

season I in the first year study. In season I, the moisture content in the salt of

control pond was 5.78 ± 0.059% and the salt from the experimental pond was 8.20

± 0.611%. The statistical analysis of ‘t’ test was made for moisture content

between the salt samples in the control and experimental pond were statistically

non-significant (t = – 2.950007; P < 0.05). In season II, the moisture content of the

salt samples analysed from control pond was 6.28 ± 0.574% and 5.72 ± 0.634% in

the experimental pond. The ‘t’ test made on the presence of moisture content

between the salt samples in the control and experimental ponds were statistically

non-significant (t = 1.24544; P < 0.05).

During season III, the moisture content was 8.46 ± 0.099% and 6.02 ±

0.577% in the control and experimental pond respectively. The ‘t’ test period for

the moisture content between the salt samples of control and experimental ponds

were statistically more significant (t = 6.65502; P < 0.05). Similarly during season

IV, the moisture content of control salt was 7.40 ± 0.033% and in experimental

pond it was 5.28 ± 0.530%. The statistical analysis of ‘t’ test shows that t-value

was 6.93569 and P-value was less than 0.05 which indicates that there was a

significant difference in the presence of moisture content between the salt samples

harvested from the crystallizer control pond and the experimental pond.

5.3.2.2. Insoluble matter (%)

Insoluble matter in the salt samples during the first study period showed a

slight increase in season I and III. But it was decreased in season II at the same

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time there was no change in between the observed samples during season IV. In

season I, the insoluble matter in the control pond salt was 0.45 ± 0.145% and the

salt from the experimental pond was 0.51 ± 0.013%. The ‘t’ test made on the

presence of insoluble matter between control and experimental ponds were

statistically non-significant (t = – 1.481226; P < 0.05). In season II, the insoluble

matter in the control salt was 0.46 ± 0.066% and 0.43 ± 0.015% was observed in

the experimental pond. The statistical analysis (‘t’ test) made on the presence of

insoluble matter between control and experimental ponds were statistically

non-significant (t = 0.355617; P < 0.05). During season III, the insoluble matter in

the control salt was 0.43 ± 0.005% and it was 0.59 ± 0.076% in the experimental

pond. The statistical analysis of ‘t’ test conducted for the presence of insoluble

matter between control and experimental ponds were statistically non-significant

(t = – 4.90098; P < 0.05). In season IV, the insoluble matter of control salt was

0.46 ± 0.031% and this same value (0.46 ± 0.024%) also observed in the salt

sample taken from the experimental pond. The ‘t’ test conducted in the presence of

insoluble matter between control and experimental ponds were statistically

non-significant (t = – 0.111111; P < 0.05).

There was a gradual decrease of insoluble matter from control to

experimental ponds during the second year study was observed. The percentage of

insoluble matter present in the salt samples of season I showed 0.53 ± 0.039% in

the control and it was 0.48 ± 0.054% in the experimental pond. The statistical

analysis of ‘t’ test made on the presence of insoluble matter between control and

experimental pond were statistically significant (t = 4.443677; P < 0.05).

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In season II, the control had 0.52 ± 0.07% and the experimental pond

showed the insoluble matter of 0.47 ± 0.043%. The ‘t’ test analysis for the

insoluble matter of the salt samples of the two ponds were statistically

non-significant (t = 2.333333; P < 0.05). During season III, the insoluble matter of

the salt sample was 0.50 ± 0.083% and 0.47 ± 0.002% in the control and

experimental pond respectively. The statistical analysis made on the insoluble

matter of the two ponds were non-significant (t = 0.560991; P < 0.05). In season

IV, the insoluble matter in the control and the experimental pond were

0.58 ± 0.103% and 0.49 ± 0.045% respectively. The ‘t’ test showed the insoluble

matter of the salt samples of the two ponds were statistically significant

(t = 0.002193; P < 0.05).

5.3.2.3. Sulphate content (%)

An increased level of sulphate from the control towards the experimental in

all the three seasons (I, II and IV) except season II was observed during the first

year study. In season I, the sulphate content of control sample was 0.83 ± 0.007%

and the salt from the experimental pond was 0.96 ± 0.016%. The statistical ‘t’ test

showed the sulphate content of the salt samples between control and the

experimental were statistically non-significant (t = – 4.444596; P < 0.05). In

season II, there was a slight decrease in the experimental sample (0.76) than the

control (0.78) was observed. The statistical analysis (‘t’ test) revealed that the

presence of sulphate content between the salt samples in the control and

experimental ponds were statistically non-significant (t = 1.028374; P < 0.05). The

sulphate content was 0.83 ± 0.075% in control sample and 1.08 ± 0.082% in the

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experimental salt sample during season III. The ‘t’ test made between the salt

samples of the control and the experimental pond were statistically non-significant

(t = – 4.930356; P < 0.05). The sulphate content of the salt samples between

control and experimental ponds was almost same in season IV and it was 1.02 and

1.06% respectively. The ‘t’ test made on the sulphate content between the ponds

(control and experimental) were statistically non-significant (t = – 4.323460;

P < 0.05).

Also the second year study the sulphate content in the samples showed a

gradual decrease from control to experimental ponds. In season I the values were

1.17 ± 0.166%, and 1.02 ± 0.130% for the control and the experimental salt

samples. The ‘t’ test analysis proved on the sulphate content of salt samples of

control and experimental pond were statistically non-significant (t = – 0.434152;

P < 0.05). Season II showed the sulphate content of the control salt was 1.53 ±

0.259% and the experimental salt was 1.04 ± 0.046. The statistical analysis of ‘t’

test showed the statistically non-significant sulphate content (t = 1.356301;

P < 0.05) between the control and experimental salt samples. During season III the

control salt sample showed the sulphate content was 1.25 ± 0.254% and 1.18 ±

0.095% was observed in the experimental salt sample. This sulphate content

between ponds were statistically non-significant (t = 1.808970; P < 0.05) proved by

the ‘t’ test. Similarly during season IV, the control sample expressed the sulphate

content was 1.23 ± 0.310% and it was 1.08 ± 0.140 % in experimental pond. The

statistical test (‘t’ test) proved there were a statistically significant (t = 4.103042;

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P < 0.05) sulphate content was noticed between the control and experimental salt

samples.

5.3.2.4. Calcium content (%)

The calcium content in the first year study showed an increase from the

control to the experimental salt samples during the seasons (II, III and IV) except

season I. In season I, the calcium content in the salt showed the decreased level

from the control towards the experimental pond. The calcium content was

0.35 ± 0.006% in control and it was 0.22 ± 0.005% in the experimental pond. The

‘t’ test expressed on the calcium content of control and experimental salt samples

were statistically non-significant (t = 2.700919; P < 0.05). But in season II, the

calcium content of the control pond was 0.20 ± 0.065% and experimental salt

sample was 0.24 ± 0.059%. The statistical analysis of ‘t’ test proved that the

calcium content between the control and the experimental salt samples were

statistically non-significant (t = – 0.8155724; P < 0.05).

Similarly, season III, the calcium content of the salt was 0.18 ± 0.081% in

the control and it was 0.23 ± 0.056% for the experimental salt samples. The

statistical test (‘t’ test) made on the presence of calcium content between the salt

samples of control and experimental ponds were statistically significant

(t = – 1.7320508; P < 0.05). Also season IV, the calcium content of the control salt

sample was 0.34 ± 0.010% and the experimental salt sample showed the calcium

content was 0.36 ± 0.008%. The analysis of ‘t’ test revealed that the calcium

content of the control and experimental salt samples were statistically

non-significant (t = – 2.98481; P < 0.05).

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The calcium content in the salt samples during the second year study period

showed significant changes in all the seasons except season II. In season I, the

calcium content of control was 0.12 ± 0.055% and the salt sample from the

experimental pond was 0.16 ± 0.081%. The analysis of ‘t’ test showed the calcium

content between the samples of control and experimental were statistically

non-significant (t = – 1.721892; P < 0.05). But in season II, the calcium content

showed a gradual increase in control than experimental and it was 0.61± 0.116%.

At the same time it was 0.18 ± 0.076% in the experimental pond. The ‘t’ test

expressed the statistical significant between the samples of control and

experimental in calcium content (t = 9.279327; P < 0.05). During season III, the

calcium content attained 0.21 ± 0.074% in control salt sample and reached

0.32 ± 0.056% in experimental salt. The analysis of ‘t’ test proved the statistically

non-significant (t = – 3.291781; P < 0.05) calcium content present in the salt

samples taken from control and experimental ponds. Season IV expressed the

calcium content of control pond was 0.30 ± 0.095% and the experimental pond was

0.32 ± 0.129%. The analysis of ‘t’ test made on the presence of calcium content

between the salt samples of control and experimental were statistically

non-significant (t = – 0.380878; P > 0.05).

5.3.2.5. Magnesium content (%)

The magnesium content in the salt samples during the first investigation

period was showed the gradual increase from the control to the experimental ponds

in the season I and III. It was 0.11 ± 0.044% and 0.18 ± 0.073% calcium content in

control and experimental ponds during season I. In season III it was 0.14 ± 0.055%

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and 0.24 ± 0.049% in the control and experimental ponds respectively. The ‘t’ test

stated that the magnesium content of the salt samples in the control and

experimental ponds were statistically non-significant (t = – 1.463850; P < 0.05) in

season I and season III (t = – 5.2654795; P < 0.05). Against this, the magnesium

content of the salt samples registered a decrease from the control to experimental

pond was 0.08 ± 0.002% and 0.05 ± 0.002% in season II. The ‘t’ test showed the

magnesium content of the salt samples of control and experimental were

statistically non-significant (t = 1.176697; P < 0.05). But during season IV, the

magnesium content of the control salt was 0.15 ± 0.074% and there was no change

in the experimental salt sample too, it was 0.15 ± 0.061%. The statistical analysis

(‘t’ test) expressed the statically non-significant (t = – 1.7320508; P < 0.05) value

of magnesium content of the salt samples between the ponds.

The magnesium content present in the salt samples was observed during the

second study period. A gradual decrease of magnesium content from control to

experimental ponds was observed in the seasons except season II. The magnesium

content in the control pond was 0.84 ± 0.051% and the salt from experimental pond

was 0.65 ± 0.103% in season I. The ‘t’ test conducted for the magnesium content

showed statistically significant (t = 18.190172; P < 0.05) between the samples of

control and experimental ponds. Season II showed the magnesium content in the

control salt was 0.51 ± 0.05% and the experimental salt sample was 0.53 ± 0.079%.

The statistical ‘t’ test for the magnesium content of the salt samples of control and

experimental ponds were statistically non-significant (t = – 0.373024; P < 0.05).

During season III the magnesium content in the salt was 0.77 ± 0.096% in control

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and 0.58 ± 0.091% in experimental pond. The ‘t’ test made on the presence of

magnesium content between the salt samples in the control and experimental ponds

were statistically significant (t = 7.8922833; P < 0.05). Similarly season IV, the

magnesium content of control salt and experimental salt were 0.64 ± 0.097% and

0.51 ± 0.080%. The analysis of ‘t’ test expressed the statistically non-significant of

magnesium content between control and experimental salt samples (t = 1.3523000;

P < 0.05) were noticed.

5.3.2.6. Sodium chloride (%)

The sodium chloride content in the salt samples of control and experimental

ponds during the first year study showed the increased level from the control

towards the experimental ponds in all the four seasons. In season I, it was

91.96 ± 0.470 in control and 94.64 ± 0.125% in experimental ponds. Similarly in

other seasons, it was 92.06 ± 0.705 and 95.22 ± 0.632% in season II, 93.86 ± 0.473

and 94.41 ± 0.766% was in season III and it was 93.10 ± 1.147% in control and

94.73 ± 1.30% in experimental on season IV. The ‘t’ test made on the sodium

chloride content in these two ponds were statistically non-significant for all the

seasons (t = – 13.308814 in season I, t = – 4.3149558 in season II, t = – 1.7040266

in season III and t = – 13.4065747 for season IV with P < 0.05).

As the same as first year study of the sodium chloride the salt samples from

the control and experimental ponds had the increased level from the control

towards the experimental ponds in all seasons. In season I it was 89.80 ± 1.308 and

93.99 ± 0.739% in control and experimental samples. Similarly in season II, 91.47

± 1.096% was observed in control but a slight increase (92.67 ± 0.657%) was

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present in the experimental pond. Season III expressed not much more variations

in the control and experimental salt samples. It was 92.13 ± 0.674% in control and

92.43 ± 0.725% in experimental. During season IV, the sodium chloride content of

control salt was 89.32 ± 0.668% and was 90.72 ± 1.018% in the experimental pond

salt sample. The statistical analysis (‘t’ test) made on the presence of the sodium

chloride content between the salt samples in the control and experimental ponds

were statistically non-significant in all the seasons (t = – 4.9681992; P< 0.05;

t = – 7.1968546; P < 0.05; t = – 2.7185765; P < 0.05 and t = – 2.8128475;

P < 0.05). It also revealed that the quality of salt was more pure in the ponds which

contains large amount of Dunaliella than the control.

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5.4. DISCUSSION

NaCl being consumed by human being from long time age is one of the

most important existing electrolytes in inter-cellular liquids of the body. The

quality of NaCl has always been an important issue because of its daily

consumption and also its application in food industries. There is variety of

methods to increase the quality of salt crystallization is one of the most common

industrial methods to prepare food grade sodium chloride (Ahmadpanah et al.,

2007). At the crystallization process, if the crystallization speed is too fast, part of

brine will be sealed in the salt crystals, resulting in more water-soluble impurities.

The most common impurities is pan salt, apart from dust are the sulphates of

sodium and calcium.

The crystallizers are the heart of the saltworks. They should give maximum

yield, best quality salt with minimum brine consumption. It has its own importance.

It is necessary that for proper control of quality, in addition checking of specific

gravity/density. The calcium and magnesium should also be determined, before

changing the brine to crystallizers (Jhala, 2006). Temperature increases the rate of

evaporation and hence causes faster precipitation of the halite crystals. The

biological systems of the solar saltworks, through its diversity and multiplicity of

functions are essential for the production of good quality salt (Davis, 2000a;

Evangelopoulos et al., 2006). In the crystallizer ponds where halite precipitates,

benthic microbial mats are usually absent (Oren, 2009a). The biological processes

of microalgae in the evaporators or the crystallizer ponds may be responsible for

the differences in salt quality. Microalgae Dunaliella salina, Coccochloris present

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in the crystallizer pond played an important role in quality salt production. It will

reduce the moisture, insoluble matter, sulphate, calcium and magnesium content in

the salt thereby improved the salt quality (Reginald and Banu, 2009). Dunaliella

salina release organic carbon under different conditions also improves the salt

quality (Giordano et al., 1994). It cleans the brine from organic substances

resulting in growth of large, glass clear salt crystals. Also the earlier evaporation

ponds contain dense communities especially in the benthic mats and gypsum crusts.

The gypsum deposit appears only in basins where basins are about 3.5 times more

concentrated than the initial seawater. As salinity increases gypsum deposition

begins as thin, discontinuous crusts and individual crystals of calcium sulphate

interspersed in the uppermost layer of the benthic communities and proceeds to

develop firm sheets (Davis, 2009). The primary gypsum crystals become

progressively heavier and flow downwards under the effect of their own weight.

Because of the importance of water thickness, the saturation is not total and

therefore crystals, initially formed, dissolve themselves by they reach the bottom of

the basin (Amdouni, 2006). Gypsum deposition occurs not only as firm to soft

sheets on pond floors, but also as powdery layers of individual microscopic crystals

which are carried downstream. The precipitation of the halite occurs only when the

solution becomes almost ten times more concentrated than the initial seawater

(Plate 5). The layers have been formed graduate from hard (at bottom) to soft (at

the top). The layer at the bottom which is the hardest salt layer is called the

“wooden layer” (Mustafa and Ali, 2010).

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The salt harvested in the constructed pan was white, creamy with very little

shows of grey colour and composd of different sizes of salt crystals. The solid

impurities (insoluble matter) are generally represented by the fine sedimentary and

organic particles and brought by wind or/and pulled out to the substratum of

crystallizers during the harvest of salt. The deposit conditions play a decisive role

in the kinetics of crystallization and thus control the degree of purity of the sodium

chloride (Amdouni, 2009). Contamination occurred by the accidentally excavated

soil or the soil surrounding the saltpans, which mixed with the salt water during the

processes of the saltpans (Mustafa and Ali, 2010). The flow of brine from pond to

pond was controlled so as to effect selective crystallization of the different

compounds such as calcium and magnesium salts found in brine in addition to

sodium chloride. Such a selective crystallization produces a better quality common

salt (Mensah and Bayitse, 2006) which are harvested whenever a sufficiently thick

layer has accumulated on the bottom. Purity depends on the type of salt. Mainly

the salt production based on an analysis of climatic factors such as humidity, wind

speed, temperature, rainfall pattern and the characteristics of soil of the area. The

annual production and quality of solar salt are influenced significantly with climatic

condition (Zhiling and Guangyu, 2009). Evaporation and precipitation of low and

high solubility salts were intimately linked to biological processes that occur in

every pond of a solar saltworks. The production of high quality salt from a

seasonal solar saltworks depends mainly on the functioning of the brine biological

system. This can be controlled by applying the correct management practices

assuming that the physical and biological information is continuously gathered and

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appropriately displayed on the ecological status of the brine system (Kavakli et al.,

2006).

Biological attributes also benefit the production of salt by removing soluble

metals, nutrients and total suspended solids from the brine and depositing them into

the sediments. A salt field with the biology in equilibrium will produce good sized

solid crystals with low impurities exploiting evaporation to its greatest potential.

Hydro-biological activities and their management were essential for the production

of high quality and quantity salt (Rahaman and Jeyalakshmi, 2009a). Algal blooms

induced by natural availability of organic or inorganic nutrients are beneficial since

they aid in increased absorption of solar heat resulting in faster evaporation which

in turn increases the salt quantity. It is also essential that they are metabolized in

time, if not as excretory products are in decomposed state, the algae can act as

chemical traps and consequently prevent early precipitation of gypsum (Davis,

1979) contaminating the sodium chloride crystals which reduces the salt quality.

Furthermore the organic impurities can contaminate and induce formation of higher

quantities of small crystals. The contaminants give half-white colour to the salt,

and it cannot be sold easily in markets. So such salts were not covered by tarpaulin

during rainy season. Instead it is purified by the rain water and then sold in the

markets. This process is time-consuming and provides less profit. High water

viscosities may inhibit salt crystal formation and precipitation whereas formation of

larger glass clear salt crystals occurs at lower levels. Benthic cyanobacterial mats

have a similar function in saltern ponds of intermediate salinity (Oren, 2000). The

production of the high quality of sodium chloride from a seasonal solar saltworks

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depends mainly on a functioning of the brine biological system. This can be

controlled by applying the correct management practices assuming that physical

and biological information if continuously gathered and appropriately displayed on

the ecological status of the brine system (Kavakli et al., 2006). Thus in a saltwork

producing quality product at the design capacity, a balanced biological system is

essential. Human populations that are located near to this saltworks interact directly

or indirectly with their activity mainly by the job. This saltworks administration is

hiring labour to work mainly during the harvesting of salt due to the enormous

production time.

In the present study, the moisture content of the salt harvested from the

control pond was more than the salt samples harvested from the experimental

ponds. In season III, the higher moisture level found in control than other ponds.

These variation in the moisture content clearly indicated that the climatic condition

influenced the moisture content (Sekar, 2010). Comparison with the salt

accumulated during the study period indicated that the salt harvested from the

control pond was small crystals (Plate 5j) contaminated with mud and other

unwanted materials with half-white in colour. Whereas, the salt harvested from the

experimental pond was white, creamy and large crystals in size (Plate 5k). In

general the salt is good for domestic uses and of course for various industrial uses.

This result is similar with the report of Al-Juboury et al. (2009).

The insoluble matter identified in the control pond was more than that of the

experimental ponds by which salt production is reduced (Sorgeloos et al., 1986;

Reginald, 2003). The differences in the insoluble matter between control and

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experimental ponds were mainly due to the presence of Dunaliella in the

experimental ponds. The existence of halobacterium in the brines and salts

oxidices organic matter that is determined to the salt quality (Davis, 1979). The

solid impurities (insoluble matter) are generally represented by the fine sedimentary

organic particles and brought by wind (Coleman and White, 1992) or/and pulled

out to substratum of crystallizer during the harvest of salt. They can also be like

the evaporated mineral precipitating at the same time as the halite. The abundance

of impurities, their nature and distribution within the crystalline mass mainly

depend on the chemical composition of the mother brine, the quantity of the

suspended matter and kinetics of crystal growth (Amdouni, 2009). Existence of

impurities will deteriorate the size of crystals. Dunaliella salina, whose cells

accumulate carotenoids, when grown in high salt concentrations and therefore

appear red. The red colour of the concentrated saltern pond is often regarded as a

significant contribution to the solar salt production process (Reginald et al., 2004).

The present study also confirmed that the experimental ponds inoculated with

microalgae Dunaliella salina showed good quality salt than the salt produced in the

control ponds during the study period even though there was slight variations in the

chemical composition of salt such as sulphate, calcium and magnesium in all the

four seasons of the two years study. These results are similar with the reports of

Reginald (2003). Microorganisms which are living in these extreme habitats may

act as sulphate reducers (Schneider and Hermann, 1990). There were some

considerable variations in the chemical composition and purity of salt due to the

inoculation of microalgae (Campbell, 1995). It is also revealed that the quality of

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salt was more pure in the ponds, which contains bulk of Dunaliella. The harvested

crystals in the present study were mostly layered, hollow, hopper-shaped pyramids

and they retain bacteria (Plate 5l).

The present study revealed that the experimental ponds inoculated with

microalgae D. salina showed good quality salt than the salt produced in the control

ponds. These microalgae involved in the considerable reduction of moisture,

insoluble matter, sulphate and magnesium content and enhanced the quality of salt.

Basically solar saltworks which will cover our country needs in salt and will offers

job to local people. In Puthalam saltworks condenser and crystallizer ponds have

Dunaliella in high quality, which is necessary for increasing the purity of the salt

(Plates 5d and 5e). The importance of it is analysed and learned through field

application. No doubt, if the same is used in all saltworks, the quality of salt will

be increased and can be valuably used for cooking. Finally solar saltworks are also

recognized to be unique cultural landscapes that deserve conservation and also

constitute destination for eco/agrotourism (Petanidou, 2000).