RU2115722C1 - Method of halobacterium biomass preparing - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: method is carried out by continuous feeding nutrient medium and constant culturing medium volume and continuous suspension removing that contains halophilic bacterium biomass. Concentration of dissolved oxygen during the process is maintained at the level not above 10-15% of saturated value. Spent medium is concentrated and recovered (partially or completely) to the culturing process. EFFECT: increased yield of biomass, increased accumulation of biologically active substances in biomass. 2 cl

Description

 The present invention relates to the medical and microbiological industry and relates to methods for producing bacterial biomass of halophilic bacteria, in particular those producing bacteriorhodopsin.

 The closest technical solution to the proposed invention is a method of culturing halophilic bacteria - producers of bacteriorhodopsin according to ed. St. USSR N 626583, priority 1977. The method is as follows.

The culture of halophilic bacteria is grown in a 6-liter flat cuvette with a working lid of 3 l at a temperature of 38 ^o C, pH 7.0 - 7.4, air aeration of 1 l / min per 1 l of suspension with continuous illumination of 0.3x10 erg / sq. cm / with LB-40 lamps.

 Initially, 2.5 liters of culture medium and 0.5 liters of seed grown in flasks are poured into the cuvette. After 3 hours, a nutrient medium with seed in a ratio of 5: 1 is pumped. Mixing is carried out using aerating air. Draining the suspension in excess of the specified working amount is carried out through the drain tube at the level in the cuvette. Thereby, the culture and environment in the cell are updated. Subsequently, every three hours, the procedure for feeding the seed with the medium into the cuvette and draining the excess amount of the suspension from it is repeated. Thus, according to the copyright certificate 626583 carry out a continuous process of growing halophilic bacteria.

 The growing process is carried out while maintaining the concentration of dissolved oxygen at the level of 50 - 55% of saturation.

 The disadvantage of this method is the inefficient semi-continuous process for growing halophilic bacteria, called by the authors a continuous process. In fact, according to this method, a batch process is carried out for 3 hours, then part of the nutrient medium is replaced in the working volume of the cuvette, and the supplied fresh nutrient medium contains inoculum that is previously constantly grown in flasks to ensure the growth process in the cuvette. Thus, the process is laborious and ineffective in the yield of halophilic bacteria biomass.

 The aim of the present invention is to intensify the growth processes of halophilic bacteria and increase the efficiency of obtaining biomass of halophilic bacteria in the industrial production of both biomass itself, which has independent use, and bacteriorhodopsin isolated from it.

 This goal is achieved in that the biomass of halobacteria is obtained by growing them under aeration and lighting conditions on a medium containing sources of carbon, nitrogen, phosphorus and mineral salts in a continuous process with continuous supply of a nutrient medium with a constant volume of the growing medium and continuous selection of a suspension containing biomass halophilic bacteria. The suspension is concentrated, in particular, on centrifugal separators, and the spent growing medium obtained by thickening is partially or completely returned to the growing process together with fresh nutrient medium, which ensures a balanced composition of the mixture fed to the cuvette. To increase the efficiency of using the mass transfer capabilities used for growing apparatuses, the process of growing halophilic bacteria is carried out in the mode of maximum use of the mass transfer capabilities of the apparatus used for growing, determined by the mass transfer of oxygen from the gas phase to the growing medium, i.e. at a concentration of dissolved oxygen not exceeding 10 - 15% of the saturation concentration. Lighting suspension is maintained at a level of 0.1 - 0.5 erg / sq.cm / s.

 The invention is illustrated by examples, not limiting the scope of the proposed method.

 Example 1

A halophilic bacteria culture Halobacterium halobium strain 353P was grown in a flat 18-cell cuvette with 15 L filling. The composition of the nutrient medium per 1 l, g:
Sodium Chloride - 250
Magnesium Sulfate - 20
Potassium Chloride - 2
Sodium Citrate - 3
Peptone - 5
Yeast extract - 2
The temperature of cultivation 38 - 40 ^o C; The pH of the growth medium 7.2 to 7.4 was maintained by feeding 2 N. caustic soda solution.

 Aeration and mixing of the growth medium was carried out by air supplied in an amount of 0.3 - 1.0 l / l / min. Lighting with LB-40 lamps with an intensity of 0.1 - 0.3 erg / sq.cm / s.

 A nutrient medium was poured into the cuvette, the temperature and pH of the medium were adjusted to normal, and the culture previously grown on rocking flasks was poured. For 10–12 h, the culture was grown in a batch mode, maintaining the temperature and pH of the growing medium at a constant level with continuous aeration and lighting. After this, a nutrient medium of the indicated composition was continuously supplied in an amount of 1 l / h and a suspension containing a biomass of halophilic bacteria in an amount of 1 l / h was continuously selected. The biomass concentration during the continuous process is maintained at a constant level for the required time, for example, for 10 days. The amount of air supplied was set in such a way as to maintain the concentration of dissolved oxygen measured by the dissolved oxygen sensor at a level of 10-15% of saturation.

 The implementation of the process with the maximum use of the mass transfer capabilities of the used apparatus for growing halobacteria allows to reduce energy costs for the process.

 The suspension was concentrated in a known manner, obtaining a yield of the condensed suspension in the form of a paste in an amount of 2.5 to 2.8 g with 1 l of the original suspension.

 Example 2

 The growing process was carried out as in example 1, however, in the preparation of the nutrient medium used culture fluid obtained by thickening the suspension in an amount of 50% of the total amount of medium supplied or 2/3 of the medium supplied. Those. the nutrient medium was prepared, as in the previous example, with a corresponding increase in its composition of peptone and yeast extract (2 or 3 times, respectively) and before being fed into the continuous cultivation process, they were mixed with the spent culture medium in a ratio of 1: 1 and 1: 2, respectively .

 Technological process indicators were maintained at the same level as in example 1.

 Reuse of the used culture medium can reduce the consumption of chemicals and reduce the amount of effluent.

 Thus, the above examples illustrate the proposed method for producing biomass of halophilic bacteria, but do not limit it.

Claims (2)

 1. The method of producing biomass of halophilic bacteria when growing them under conditions of aeration and lighting on a nutrient medium containing sources of carbon, nitrogen, phosphorus and mineral salts and subsequent concentration, characterized in that the growing process is carried out with a continuous supply of nutrient medium and continuous selection of the suspension, containing biomass of bacteria, while the concentration of dissolved oxygen is maintained at a level not exceeding 10 - 15% of saturation.  2. The method according to claim 1, characterized in that the selected nutrient medium after concentration is partially or completely returned to the growing process together with fresh nutrient medium.