LIGNITE HUMIC ACID FOR ENHANCED HATCHERY SHRIMP SEED PRODUCTION
Field of the invention
The present invention relates to the application of humic acid in aquaculture. In particular, the present invention relates to the application of humic acid produced from the lignite for hatchery shrimp seed production. The present invention further relates to the lignite based humic acid is used as a growth promoter for phytoplankton. The shrimp fed with the humic acid supplemented phytoplankton exhibited improved growth and survival rate.
Background of the invention
Phytoplankton are the first link in the food chain and are known as primary producers, which is consumed by zooplankton and other small animals in the marine environment. Aquaculture industry use cultured phytoplankton as a primary food source for the cultivable organisms. The widespread use of phytoplankton as feed finds a struggle of high production cost in mass culture systems due to the usage of fertilizers and other chemicals. In alternate, some of the studies reported that the humic acid was used as a feed supplement to increase the growth of phytoplankton. Humic acid is a major constituent present mostly in soil, peat and coal. It is formed by biodegradation of organic matter and serves as natural organic fertilizer. It has been commonly used as a soil supplement in agriculture, and less commonly as a human nutritional supplement. The prior arts related to this study are as follows as
Reference may be made to the Chinese patent (CN101863705B), wherein preparation of growth promoter for aquaculture which includes the plant hormone gibberellins, salts, bacillus powder and sodium humate.
Prakash and Rashid (Limnology and Oceanography 13, 598 - 606, 1968) examined the influence of humic substances on the growth of marine phytoplankton, dinoflagellates. The

humic acid used in the study was extracted from the soil and marine sediments. The authors found that the humic acid was more active than fiilvic acid and growth of marine phytoplankton is concentration dependent on both cases.
Prakash et al. (Limnology and Oceanography 18, 516 - 524, 1973) investigated the influence of humic substances extracted from the decomposed residues of marine algae and mangrove leachates on the growth of marine phytoplankton, diatoms. The humic acid stimulates the growth of marine diatoms which is dependent on concentration and molecular size of humic acid.
Carlsson et al. (Marine ecology progress seriesl27, 213-221, 1995) studied the effect of addition of humic substances derived from river water on coastal phytoplankton community. The Phytoplankton biomass was slightly higher in the humic treatment compared with the non-humic treatment. The growth of dinoflagellates and small flagellates was enhanced by humic addition.
Martin et al. (Journal of the Marine Biological Association of the United Kingdom, 1-12, 2014) reported that adding low concentrations of natural humic substances to the algae cultures resulted in enhanced growth rates compared to the control experiments which possibly due to the increase in the bioavailability of trace metals or other nutrients.
The drawbacks associated with the prior arts are as follows: The humic acid used in the prior studies was prepared from the sources such as soil, river water, decomposed residues, marine sediments and sub-bitminous coal. Moreover, the existing studies focused the effect of humic acid on growth of marine phytoplankton only.
Therefore, keeping in the view of drawbacks, the inventors of the present invention examined the effect of low grade coal, lignite based humic acid (supplied by Neyveli Lignite

Corporation India Limited, Neyveli, Tamil Nadu, India; Patent No D-CHE/0886) on growth of fresh water phytoplankton as well as marine phytoplankton.
Objects of the invention
The main objective of the invention is to propose a growth supplement for phytoplankton to enhance the biomass production
Another object of the invention is shrimp feed which contains fresh water phytoplankton grown in a medium containing lignite based humic acid.
Yet another object of the invention is shrimp feed which contains marine phytoplankton grown in a medium containing lignite based humic acid.
Still another object of the invention is the feed for shrimp which promotes the growth and cost effective.
Detailed description of the invention
Culturing of phytoplankton
According to the invention, algae such as Nannochloropsis sp., Tetraselmis sp., Dunaliella salina and Spirulina sp., were grown in medium containing lignite based humic acid. The algae were cultured by inoculating 3 ml of respective mother culture in 250 ml Erlenmeyer flasks containing 100ml of sterilized growth medium and humic acid of different concentration ranging from 100 ul to 300 ul. The sterilized growth medium consist sodium nitrate, sodium dihydrogen phosphate and sodium silicate, trace metal solution and vitamin solution. The temperature of flasks was maintained in the range of 16 to 27°C, salinity of 34 to 39ppt, pH in the range of 7.0 to 9.0. The light intensity was maintained in the range of 2300-34001ux with photoperiod of 12h light and 12h dark condition. The flasks were agitated

occasionally. Table 1 shows that the effect of humic acid at different concentration on growth of various algae.
Evaluation of phvtoplankton on shrimp growth:
Healthy shrimp Litopenaeus vannamei of different stages from Zoea stagel to post larval stagel were cultured in tanks with seawater. The sea water was filtered using lum filter and sterilized by autoclaving or pasteurization. The salinity of sea water is 37ppt and temperature maintained is 27±1°C under continuous aeration. The shrimp was fed for 4 times/day on every 6hour interval. The shrimp was fed with Thalassiosirapseudonana with cell density of 25000 - 30000 cells/ml and artemia at different concentration depends on the stage of animal. The phvtoplankton feed was given 4 times/day on every 6hr interval. In addition to phytoplankton feed, shrimp was fed with Exorich (lppm) and Vitamins (lppm). The survival rate and total length of the shrimp was calculated. The shrimp feed with the humic acid supplemented phytoplankton (experimental) shows comparable growth (Table 2) and survival rate (Table 3) to the phytoplankton grown in medium without humic acid (control).