APPLICATION OF BIOSURFACTANTS IN WASTEWATER BIOFILTRATION TECHNOLOGY USING DAPHNIA MAGNA

Introduction. The problem of minimizing organic pollution from agricultural enterprises, including aquaculture farms, is an incentive for the development of bioremediation research. Among the various methods of wastewater treatment, along with traditional physicochemical methods, biological treatment technologies are actively used, mostly using aerotanks with activated sludge (Sabliy, 2013). Along with this, when solving water purification problems, including for the needs of industrial fish farming, biofiltration methods with the participation of planktonic organisms, in particular, branchial crustaceans, deserve special attention.

A specific Daphnia feeding filtration apparatus can provide highly efficient wastewater treatment of suspended fine particles that have a low sedimentation rate and are poorly retained on mechanical filters (Pau et al., 2013; Serra et al., 2022). An important advantage of this method of water purification is the possibility of using the obtained zooplankton biomass as live food for fish farming (Khudyi et al., 2016; Hyman et al., 2021). The filtration properties of zooplankton can be increased by growing it in the presence of biosurfactants – surface-active substances of biological origin. Biosurfactants are synthesized by microorganisms, they are low-toxic compared to chemical surfactants and biodegradable (Mir et al., 2017). In addition, the use of biosurfactants allows to increase the assimilation of nutrients by cladocerans and intensify the growth of their biomass.

A promising surfactant producer is Rhodococcus erythropolis, which is capable of synthesizing trehalosolipid surfactants (Koretska et al., 2018). These microorganisms are used in bioremediation technologies, as they provide biodegradation of toluene, naphthalene, biphenyl and other cyclic organic compounds (Dockyu et al., 2018). However, the use of such substances requires a preliminary biotesting procedure. Therefore, the aim of this study was to evaluate the possibility of using trehalosolipid biosurfactants for intensive cultivation of Daphnia magna culture for the purpose of biological purification of water bodies.

Materials and methods.

The maintenance of the laboratory culture of Daphnia magna was carried out in a climatic chamber with a stable temperature regime of +20±1°C and a light day duration of 16 hours. The acute toxicity level indicators were determined for different concentrations of the trehalozolipid biosurfactant preparation obtained from the culture fluid of Rhodococcus erythropolis AU-1 in the Department of Physical Chemistry of Fuel Fossils of the L.M. Lytvynenko Institute of Physical Organic Chemistry and Coal Chemistry of the NAS of Ukraine.

The initial density of Daphnia culture was 100 individuals/l. Dead and living individuals were counted during the first hour of the study, then after 24 and 48 hours of the experiment using a Bogorov counting chamber under a binocular microscope MBS-9. LC50 determination for 24 and 48 hours was performed using probit analysis (DSTU 4173:2003, 2004). During the experiment, animals were not fed.

To intensify the growth of D. magna biomass, cultivation media were prepared with a concentration of trehalozolipid preparation of 1 mg/l, 2 mg/l and 3 mg/l. Daphnia cultures were used as controls, in which there was no biosurfactant in the growing medium. Water from a mechanical filter of a recirculation system for growing fish, into which Daphnia was placed, was used as waste water. The study was conducted for 4 days. To assess the efficiency of treatment, the optical density of waste water was determined on a KFK-2 photoelectrocolorimeter (530 nm), pH, electrical conductivity, total mineralization and dissolved oxygen content in water were determined using an instrument for determining the physicochemical parameters of water IR67 Combo. The results obtained were statistically calculated using the Student’s t-test.

Results and their discussion.

The results of the analysis to determine the acute toxicity of trehalozolipids using Daphnia magna test cultures showed a rather low level of toxicity of the studied substances. Thus, the minimum concentration of trehalozolipids at which the death of Daphnia was observed within 24 hours of exposure was about 300 mg/l, and the concentration at which half of the individuals of the test culture died within 24 hours was 479 mg/l (Fig. 1).

Fig. 1. Dependence of the mortality rate of Daphnia magna within 24 hours under the influence of different concentrations of trehalosolipids (A) and their LC50 (B)

When incubation was increased to 48 hours, the LC50 decreased by only 27% compared to the LC50 for 24 hours and amounted to 347 mg/l (Fig. 2). At trehalozolipid concentrations up to 250 mg/l, no death of daphnia was observed within 48 hours.

Trehalozolipid biosurfactants exhibit extremely high toxicity, with 100% of test specimens dying within 24 hours, at fairly high concentrations – 600 mg/l and above. Obviously, such concentrations should not be used in aquaculture practice.

Fig. 2. Dependence of the mortality rate of Daphnia magna within 48 h under the influence of different concentrations of trehalosolipids (A) and their LC50 (B)

Taking into account these results, for the effective increase in Daphnia biomass we selected concentrations of trehalozolipid biosurfactants of 1 mg/l, 2 mg/l and 3 mg/l. It was found that the use of all three studied concentrations of trehalozolipids had a positive effect on the number of Daphnia magna individuals. Thus, the highest density of Daphnia cultures in the experimental groups was 1.3-1.7 times higher compared to the control group (Fig. 3). The maximum number of individuals was noted for Daphnia cultures exposed to trehalozolipids in the lowest of the studied concentrations – 1 mg/l. However, higher concentrations of the drug provided a faster increase in the number of individuals. In particular, at concentrations of 2 and 3 mg/l, the density of Daphnia magna cultures on the 6th day of the experiment was 1.5 times higher than in the first and control groups.

After selecting the optimal concentrations of biosurfactants, in the next stage of research, water from the mechanical filter of the experimental recirculation system of the Yuri Fedkovych National University of Chernivtsi was used to evaluate the efficiency of biofiltration of Daphnia magna. This water is saturated with organic matter due to the vital activity of fish and due to its leaching from feed. Accordingly, it can also be used as an alternative medium for cultivating feed zooplankton.

The maximum number of individuals was noted for Daphnia cultures exposed to trehalozolipids at the lowest of the studied concentrations – 1 mg/l. However, higher drug concentrations provided a faster increase in the number of individuals. In particular, at concentrations of 2 and 3 mg/l, the density of Daphnia magna cultures on the 6th day of the experiment was 1.5 times higher than in the first and control groups. After selecting the optimal concentrations of biosurfactants, at the next stage of research, water from the mechanical filter of the experimental recirculation system of the Yuri Fedkovych Chernivtsi National University was used to assess the efficiency of Daphnia magna biofiltration. This water is saturated with organic matter due to the vital activity of fish and due to its leaching from feed. Accordingly, it can also be used as an alternative medium for cultivating feed zooplankton.

It was found that Daphnia individuals actively reduce the optical density of the effluent water during a 4-day stay in it without additional feeding, which indicates the effectiveness of their use as filter media (Fig. 4). Thus, the optical density of the effluent water decreases more than twice after just one day of Daphnia staying in it, and 7 times after 4 days. The addition of biosurfactants leads to a more efficient filtration process, with the best indicators being established when using a preparation with a concentration of 1 mg/l. The results of the study of the level of total mineralization and electrical conductivity (Fig. 5) of circulating water from the recirculation system indicate a decrease in these indicators under the conditions of use of all studied concentrations of biosurfactants, which indicates the activation of the filtration properties of Daphnia under the conditions of their use. In contrast, the pH and dissolved oxygen content indicators practically do not change compared to the control.

During the cultivation of daphnia, a gradual increase in the pH of the wastewater is observed, reaching maximum values ​​(7.7 for the control, 7.5-7.6 for the experimental groups) on the 2nd day of the experiment. The dissolved oxygen content gradually decreases in both the control and the experimental groups from 6.9 mg/l to 5.3 mg/l.

Thus, the possibility of intensifying the growth of Daphnia biomass under the conditions of using the trehalozolipid biosurfactant preparation in the concentration range of 1-3 mg/l has been shown. An increase in the filtration activity of the Daphnia magna culture was noted when using the preparation with a concentration of 1 mg/l.

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