ULTRASONIC STIMULATION OF CHLORELLA POPULATION GROWTH

Ultrasonic (high frequency/low intensity) stimulation of Chlorella vulgaris is shown to intensity population growth. Toe effect is caused by a change in the cellular membrane po­ tential as a result of increased permeability of the membrane; metabolic rate is thus determined by the cell's energy demand. It is suggested that sonication can be applied in biotech­ nological processes involving Chlorella.


JNTRODUCTION
The concept of virtually waste-free production as well as certain technologies in fish processing industry make it possible to utilise raw materials almost completely, to process the by.;-products and wastes, and to reduce pollution of the environment.
Improved equipement and technologies allow to extend the scope of waste utilisation and are conducive to proposing novel applications.
BiotechnologicaLprocesses, effected by both living cells and certain biologically active substances extracted from them"(e.g., enzymes contained in the cells) provide an excellent tool with which to process industrial wastes.
The greatest potential is offered by taking advantage of processes that normally operate ,vithin living cells of microorganisms.However, a large-scale application of those processes is problematic due to their relatively low rates: To increase the rate of a biotechnological process calls for intervention into metabolic regulation.This can be effected in different ways.However, there has been so far no simple and versatile industrial-scale method whereby microorganisms could be stimulated to increase the rate of processing wastes and sewage as their substrates and to perform supersynthesis.
Increased permeability of the cytoplasmic cellular membrane is the basis of physiologi cal metabolic regulation, whereby supersynthesis is achieved (Vorobieva 1989); as a rule, this is the process that determines the rate and extent of release of substances.
Ultrasonic treatment (sonication) is one of the ways in which cytoplasmic membrane permeability can be increased, thus intensifying development of a microorganism.Ultrasonic stimulation of exchange processes in microbial cells has already been used for medical, vet erinary, and pharmaceutical purposes.Sonication applied to microbial population has a great potential for intensifying biotechnological processes.Results of studies can be viewed from a systemic level, whereby a system is regarded as an active transformer of matter and en ergy; there is no need to revert to interaction between in, dividi,ml components of the system.
From a practical point of view, ultrasonic stimulation of the microalga Chiarella vul garis populations in order to intensify their rate of sewage treatment in a fish processing plant is of a great importance.

MATERIAL AND :METHODS
Chlorella vulgaris is present under natural condition in the Tamja.In the experiment, population densities of 14xl0 6 cells mr 1 similar to the natural ones, were used.
The algae were cultured in connection with developing a complex sewage technology to be used in a fish processing plant.An ultrasonic generator (880 kHz, intensity range of 0.1?<10 4to l.0><10 4 Wt m• 2 , sonication time of l to 10 min) was used in the experiment.Algal cultures were kept in 500 cm 3 flasks; no mixing was apllied to those microcosm which were allowed a free gas exchange with the atmosphere.The temperature was kept at a stable level of 26± 1 ° C with an ultrathermostat.Intensification of biological processes in the population,was assessed from changes in population density, respiration, and rate of photosynthesis, Population density was estimated from counts made in the Blirker chamber.Rates of respiration• and photosynthesis were determined concurrently, in the light and in darkness, using an oxymeter.

RESULTS AND DISCUSSION
Analysis of the • data pertaining to the population growth dynamics of Ch. vulgaris shows changes in the course of S.cshaped curves describing the population growth following sonication.The changes may be divided into 5 stages (Fig. 1).lncrease in the sonication intensity up to 0.6x 10 4 Wt m• 2 did not affect the shape of the curve (Stage 1).A subsequent increase up Jo 0.8x10 4 Wt m• 2 brought about a shortened lag phase, but the population density remained virtually unchanged (stage 2).At the sonication intensity of l.Oxl0 4 Wt m• 2 , the lag phase was no longer shortened, the population density increased by the factor of 1. 5 and the expotential phase was elongated by a factor of 1.3 (stage 3).Following the sonica tion intensity increase to 1.2Xl0 4 Wt m" 2 the lag phase duration increased 1.8 times relative to the control, the population density increasing 1.2 times.It should be mentioned that un der such experimental conditions the system is subject to an ,,acoustic shock" which intensi fies biomass accumulation (stage 4).At the sonication intensity exceeding l.2xI0 4 Wt m• 2 , the culture was show'ing•symptoms of destruction (stage 5).
The inorganic phosphorus uptake i... ncreased during the lag phase only at stage 2. The increase continued until the expotential phase at stage 3, while a decrease during the lag phase and an increase during the exponential phase were observed at stage 4. The uptake decreased at stage 5.
Analysis of the oxygen release dynamics, regarded as a measure of gross photosynthesis rate, showed that the rate practically did not differ during stages 1 to 4 from the control rate.At stage 5, the gross photosynthesis rate plummeted throug.. hout the entire duration.
Chemical composition of the sonicated and non-sonicated populations showed the protein content in the first, while subject to the non .; destructive treatment, to increase by the factor of 3 at the intensification stage and to double at the ,,acoustic shock" stage, compared to the control.In those parts of the spectrum there is no increase in the relative intensity of partial ab sorption in the sonicated cells.On the other hand, sonication intensities leading to the ,;acoustic shock" and cell destruction produced marked differences in the spectra, more pronounced in the parts associated with the sonication intensity that causes cell destruction that in those related to the ,,acoustic shock" -producing intensities.
Analysis of different spectra showed also a decrease and an increase in absorption; ob served at 1550-1660 and at 1630 cm•1, respectively.This is evidence of changes occurring in various forms of pol yp eptid chain configuration and destruction of the protein spatial structure.
Analysis oflipid infrared spectra produced by the samples failed to detect any marked differences between various sonication intensities.Absorption was observed.to increase at 1065 cm• 1 (monoglycerides) and at 1710 cm• 1 (carbonyl compounds) under the ,,acoustic shock" treatments: absorption increased at 3560 cm• 1 and decreased at 3012 -3020 cm• 1 (unsaturated fatty acids) (Fig. 2).Similar assays ran during the daytime showed the changes in those cells subject to the ,,acoustic shock" to be almost completely absent.The cellular protein affected by the ultra sonic treatment of intensity in excess of 3000 kJ m• 2 during the daytime underwent further destruction; lipids were brought to further oxidation and hydrolysis (increase in the absorp tion intensity curve at 3560 cm-1 ).
Ultrasonic spectroscopy analyses showed that those treatments intensifying growth of the microalga produced no change in proteins and lipids of the sonicated populations of Ch. vulgaris.Changes brought about by the treatments leading to the ,,acoustic shock" are small, too, and involve partial protein denaturation and increase in the lipid peroxide group.In those treatments leading to destruction cells, irreversible denaturation of protein oc curred, followed by protein hydrolysis and oxidation and hydrolysis of lipids.
The present study shows that the Ch.vulgaris population sonicated with intensities of 0.8xl0 4 to l.Oxl0 4 Wt m-2 increased its inorganic phosphoms uptake, the increase being typical of higher ATP activity.
The sonication intensity of l.2xl0 4 Wt m-2 produces some destruction in the natural structure of protein.This is tum intensifies protein synthesis as the cell is stimulated to com pensate for the loss.Sonication seems to enhance biological sewage treatment with the use of Ch. vulgaris in fish processing plants.Sonicated and non-sonicated populations used in sewage treatment technologies showed m.arked differences 1.-,_ their nitrogen reduction ca pacities( 0 .5 and 2.0 mg/c !cm\respectively); amino acid content decreased by a factor of 1.5 (0.06 and 0.09 mg/dcm 3 , respectively) while a 2.5-fold reduction in nitrate nitrogen (0.01 and 0.1 mg/dcm 3 ) and a 3.5-fold one in nitrite nitrogen (0.08 and 0.28 mg/dcm3, respec tively) were recorded as well.
The positive effect of low intensity sonication is due to the fact that a cell affected is mobilised to use its reserves for growth and development.The effect is sufficient to affect biological processes of regulation and compensation, without demaging reversible reactions (Akopjan et al. 1988).
The effect is caused by microflows in the cell and around it and an increase in the cellu lar membrane permeability, which changes the membrane potential.
The change in membrane potential in tum accelerate$ ATP synthesis and intensifies biological processes.Destruction and reduction in mitqQJlondrial membrane affects the membrane's non-specific conductivity for }I' or produces a complete disappearance of the membrane, in which case the ATP activity has to increase.It can be concluded that a rapid and reversible ATP synthesis can take place in an active centre without energy expenditure on the part of the hydrogen ion electrochemical potential.This is the process the rate• of which increases 1000-fold once the membrane energised (Anis:imov et al. 1986), i.e., when its potential increases, which takes place following increase in cellular membrane perme ability due to sonication.Metabolic rate is controlled by the cell's energy demand rather than by contents of various substances in the medium.
Authors' address: Ph.D. Natalia Dolganova Astrakhan Technical Institute of Fisheries Astrakhan Russia Received: 1993.12.17