Production of Chlorella (Chlorophyta) biomass enriched by selenium and its use as a feed supplement

Selenium (Se) is an essential microelement which is of great importance in human and animal health. Its deficiency has been proved to contribute to the pathogenesis of diseases such as cancer, cardiovascular, auto immune or metal afflictions. In most European countries, the daily intake of Se for adult does not exceed 40.50 micro g/day, while the recommended value is 60-70 micro g. To increase it concentration in animal and consequently in human body, inorganic selenite salts are commonly added to animal feed. Nevertheless, the utilization of Se added to the organism in inorganic form is low. A technology for the large-scale production of patented Chlorella sp. strain (micro submax = 0, 18/h at culture temperature 35-37 deg C and pH 6-7.5) containing organically bound Se was proposed. The cultivation proceeds heterotrophically in fermentors, on nutrient media with urea as a nitrogen and glucose as a carbon source. The product, the spray-dried biomass of disintegrated algal cells is characterized by high level of Se, and good intake by the host organism. A two-step fed-batch culture regime was developed: the first step starts with an inoculum concentration of about 1.0 g algal dry mass per 1 L of nutrient medium containing 80 g of glucose and selenite salts. After 40h of exponential growth, a second dose of nutrients and Se is added. A concentration of more than 70g algal dry mass/L of media is attained after 55-60h of cultivation, with a mean production rate 1200-1300 mg/L algal dry mass/h and a cell yield to glucose ratio of 0.50. Sequential extraction by isopropanol/chloroform, water/buffer and sodium dodecylsulphate solution was designed for differentiating the chemical forms of Se in algal cells. Inductively coupled plasma mass spectrometry was employed for the determination of Se in individual fractions. Inorganic non-metabolized selenite was quantified in aqueous extract using separation on anion exchange resin followed by hydride-generation ICP optical emission spectrometry. Fractionation provides information on the content of Se in non-polar, protein, polar water soluble, and non-soluble organic fractions and non-metabolized selenite. The following experiments confirmed the high utilization of organic Se by host organisms: a) a group of 10 sows was fed per head and day a basal diet supplemented in the course lactation period with Chlorella biomass containing 230 micro g of Se, to the second group, 230 micro g of Se per head and day was added in the form of the sodium selenite salt. It was found that while the amount of Se in the blood of the animals supplemented with inorganic Se was 139 ng/mL, the amount of Se in the blood of the Se-Chlorella group increased up to 299.5 ng/mL and the milk production of the sows increased by 23.3%. b) while the volume of semen, motility of sperm and sperm-cell concentration in the group of breeding boars fed a basal diet decrease in the summer period, compared with the winter one, addition of 230 micro g Se in Clorella supplement increased the above values by 11-22%. c) In 27-week experiment, laying hens housed in battery cages were fed a basal diet supplemented with sodium selenite(a) and Se-enriched Chlorella(b) both in concentration of 0.3 mg Se/kg. A significantly higher laying rate and egg weight was found in Se-Chlorella variant. Egg white Se content increased from 1.36 to 2.05 and egg yolk content increased from 0.93 up to 1.60 mg/kg dry mass at Se-Chlorella compared with the Na-selenite variant.