Enhancement of Spirulina platensis Remediation Action Using Biosurfactants for Wastewater Treatment
Pollution of water systems with heavy metals is an urgent problem for many countries. Wastewater is the main pathway for pollution of rivers and water systems from heavy metals. Wastewater treatment is expensive and not always ecologically friendly. That is why alternative methods of water purification based on the use of harmless biological objects are very relevant. This work focused on the framework of a new approach of water cleansing polluted by lead and copper, by using combined action of the ecological potential of algae Spirulina platensis and chelating agent – biosurfactant. It has been chosen to work the concentration of metals – 100 ppm. It is clear from a series of tests that the adsorption of Cu2+ and Pb2+ ions by biomass of Spirulina is improved and sped up by using ecologically friendly biosurfactants—Rhamnolipids and Trehalose lipid. Specifically, the process of metals removal from polluted water is sped up by 24 h, which will be considered in creating a quick response strategy of wastewater treatment technology. Rhamnolipid1 enhances the absorption of copper by Spirulina Platensis by about 42%, Rhamnolipid2-by 68%, and Trehalose lipid by 73%. These results are more important in comparison to the well-known chelating agent of heavy metals—EDTA, which is toxic to the environment. Obtained experimental data will possible to provide an inexpensive and ecologically friendly approach for purifying wastewater from lead and copper ions.
Article Highlights
- Spirulina platensis as an ecologically friendly remediator for water purification;
- Spirulina platensis uptakes Cu2+ and Pb2+ ions from wastewater;
- Biosurfactants—enhancer of the dynamics of heavy metals absorption by Spirulina.
Introduction
Nowadays, heavy metal pollution of terrestrial and abovewater ecosystems has become a global environmental challenge. The gross violation of natural ecological equilibrium and ecosystems-ground and surface waters get an irreversible reaction, reflecting on the local population’s health (Briffa et al. 2020). The sources of water pollution are urban, agricultural, and industrial wastewater. Heavy metals occur in ecosystems from prolonged exploitation of ore deposits. It creates serious problems with environmental protection issues (Tumanyan et al. 2020). Excess amounts of heavy metals have a toxic effect on the health of people. This is often the cause of mental disorders in children (Jaishankaret al. 2014). Too much copper causes physiological and proteomic changes in plants (Ahsan et al. 2006). In the human body, copper accumulates in the liver, which leads to liver failure and metabolic disorders – Wilson’s disease. It causes skin pigmentation, low energy, and chronic fatigue. An enormous amount of copper, as well as lead, causes the most severe complications of the human nervous system (Thomson 2006; Zischka 2014).
Recently, is a priority to introduce technologies that are characterized by low costs and efficiency against a wide range of pollutants (Verma and Suthar 2015; Kurniawan et al. 2006). Bioremediation is a flexible technology based on the unique ability of organisms to cleanse any contaminated environment. The advantages are technological feasibility, low costs, minimal rainfall, and competitive performance (Kurashvili et al. 2018; Prasad 2003).
Since the early 2000s, researchers from many countries involved purify wastewater from heavy metals using different algae (Daud et al.2018; Lin et al. 2020; Romera et al. 2007; Sekomo et al. 2012; Verma and Suthar 2015; Zeraatkar et al. 2016), bacteria and fungi (Chaturvedi et al. 2015; Kim et al. 2015). Enterobacter cloacae strains can absorb almost 0.03% of the copper from the polluted area. Micrococcus luteus DE2008 is considered a microorganism capable of regenerating lead (sorption 0.7%) and copper (sorption 0.5%) of contaminated environments (Puyenet al. 2012). Due to World Health Organization, Lead is a cumulative toxicant that affects multiple body systems and is harmful to young children. Lead in the body is distributed to the brain, liver, kidney, and bones. It is stored in the teeth and bones, where it accumulates (WHO 2019).
Cyanobacteria are the most suitable bio sorbents and bio accumulators because of their wide distribution and flexible metabolism. They are the best detoxifiers (Cepoi et al. 2016). Arthrospira platensis grows well in both saltwater and alkaline environments (Kumar et al. 2020). It has a well known Spirulina as beneficial to human health for a long time, as it releases heavy metals from the liver, gastrointestinal tract, and reproductive system (Bhattacharya 2020).
During the past 10 years, many studies have shown that Spirulina purifies water contaminated with chemical pollutants. A recent study in a river (Yamuna) clarifies the ability of algae to remove heavy metals (Cu, Cd, Ni, Cr, Pb) from water (Kumar et al. 2020). Heavy metals affect produce of Spirulina biomass. Copper affects microorganisms. It Disrupts cellular function and inhibits enzyme activities (Dixit et al. 2015; Nagajyoti et al. 2010; Salem et al. 2000).
Based on experiments conducted at the Georgian Agrarian University, Spirulina showed resistance to pollutants, while heavy metals did not affect its biomass and chlorophyll production. Arthrospira platensis showed high efficiency in treating copper-contaminated water. The physiological parameters of Spirulina under the influence of copper on algae were studied in the 100 ppm concentration of Cu2+ ions inhibition of biomass cumulation by 15% and decline of chlorophyll content by 30% (Tabagari et al. 2020). With lead, it denatures nucleic acid and protein, inhibits enzymes activities and transcription (Fashola et al. 2016; Nagajyoti et al. 2010). We conducted experiments to investigate the physiological properties of Spirulina in an environment contaminated with copper and lead. According to the experimental results, the mentioned heavy metals with apply of 100 ppm did not change the physiological parameters of Arthrospira platensis.
Our goal is to intervene to improve the biosorption of heavy metals by Spirulina in less time. The study we presented is new and has never been researched.
Observe on the influence of chelators on the ability of Spirulina we have started with EDTA as a classic cheating agent. We have determined the absorbed quantity of heavy metals by atomic absorption spectroscopy. As a result, add EDTA to the environment contaminated with copper increased the absorption capacity of the Arthrospira platensis, and with lead, on the contrary, decreased it.
Having outcomes of EDTA, we continued our research on biosurfactants. These compounds have been considered a “green” product with renewable resources (Elis acirc ngela et al. 2015). Complexes of heavy metals with EDTA are much more tenacious and not biodegradable (Grčman et al. 2001).
Surfactants are amphiphilic molecules able to reduce the surface tension between two unmixable phases (AguirreRamirez et al. 2021; Otzen 2017). Many bacteria create biosurfactants during the grown-on water substrates. Biosurfactants are forceful under extreme temperature, pH, and saltiness (Kumar and Das, 2018). The biosurfactants’ ability to increase cell membrane permeability helps enhance various biologically active preparations (Lubenets et al. 2013). Most overall, biosurfactants are glycolipids. Microbial glycolipids are four fundamental groups: Rhamnolipids, Trehalose lipids, Sophorolipids, and Mannosylerythritol lipids. To form a better view of the influence of biosurfactants, we examined three kinds of biosurfactants—Rhamnolipid 1 (RL1) and Rhamnolipid (RL2), and Trehalose lipid (TRL). As the name suggests, Rhamnolipids contain a Rhamnose unit or units linked to a 3-hydroxyl fatty acid unit or units across the β-glycosidic bond. Rhamnolipids are monoRhamnolipids and di-Rhamnolipids, depending on the number of Rhamnose units in the molecule (Tiso et al. 2017). Trehalose lipids and Rhamnolipids can speed up biomass production (Koretska et al. 2020), so we hypothesize that because of this ability, the amount of metal absorbed will improve after they are added. This ability of Rhamnolipids and Trehalose lipids has inspired us to raise Spirulina capacity to absorb copper ions. We evaluated the consequences, and all three biosurfactants had different effects on Arthrospira platensis capabilities. As a result, TRL worked best. In particular, it increased the maximum amount of copper ions absorbed by 73%, RL2 boosted by 68% and RL1- by 42%.
Based on these data, it is possible in the future to apply model tests of the technology in water contaminated with copper.
Materials and Methods
The Influence of Cu2+and Pb2+ ions on the Formation of Biomass and Chlorophyll of Spirulina
The biomass of Spirulina platensis has been got via cultivation in standard Zarrouk’s medium (pH–8.7; content in g/L: NaHCO3 – 16.8, K2HPO4 – 0.5, NaNO3 – 2.5, K2SO4 – 1.0, NaCl – 1.0, MgSO4·7H2O – 0.2, CaCl2·2H2O – 0.04, FeSO47H2O – 0.01, EDTA – 0.08; and microelements kit A5 – 1 mL). The incubation was carried out with permanent air barbotage (rate of airflow 2 L/min), at temperature 25 °C, and under following illumination conditions: a photoperiod of lighting 16L/8D (16 h of light: 8 h of dark), a total photosynthetic photon flux density (PPFD) of » 15 μmol m−2 s−1.
From many experiments on the selection of the maximum concentration of metals, at which there are no significant changes in the physiologically important parameters of Spirulina, the formation of biomass and the production of chlorophyll, a concentration of 100 ppm of metal ions was chosen (100 ppm consequent to 100 mg/L metal ions). The presented studies were carried out at concentrations of 100 ppm for both metals—copper and lead. Experiments were provided in the glass tanks (vol. 20 L). For cultivation of Spirulina in Cu2+-containing medium were chosen CuSO4 and as Pb2+-containing medium was selected Pb (NO3)2. To measure the effect on biomass at the initial moment, the biomass of Arthrospira platensis was 10 g/l and the other optimal conditions were the temperature of cultivation—25 °C; Daylight illumination; duration of incubation—120 h. As for the influence on form chlorophyll when water is contaminated with heavy metals, in the control sample (without contamination) chlorophyll was 8 mg/g of fresh Spirulina biomass.
For measurement of fresh biomass productivity and chlorophyll formation by Spirulina, the following method was elaborated: the incubation medium was centrifuged at
1000 g for 20 min, and the obtained pellet was weighted. The obtained fresh biomass was treated with acetone and contains chlorophyll was determined spectrophotometrically at 652 nm according to the standard method (Arnon 1949).
The biomass of Arthrospira platensis in incubation medium has been measured spectrophotometrically at 750 nm (Butterwick et al. 1982). The chlorophyll content in the initial control solution was 8 mg/g in the fresh biomass of Spirulina. The tests on chlorophyll and biomass were carried out at 100 ppm for both metals (Lead and Copper) 1st and 2nd samples were taken after 4 and 8 h on the first day and others once a day on subsequent days.
To exclude the number of heavy metals that can pass into sediments, a test was carried out directly on the biomass of Spirulina platensis to measure Cu2+ and Pb2+ content by atomic absorption methods. Tests were carried out on artificially contaminated water with a concentration of 100 ppm of Pb2+ and 100 ppm Cu2+, respectively. Samples were taken every 4 h on the first day and once a day for the next four days. A control sample was taken before heavy metal contamination of the water. Spirulina biomass ranged from 4.0 to 4.5 g/L. Disintegrate samples were carried out according to GOST 30,178–1996 by ash method (Caroli 2006; Interstandard 2013). Air-dried samples were weighed and placed on porcelain jars. After firing the samples in a muffle furnace at 550 °C for 2–3 h, a few drops of concentrated nitric acid were added to the dry residue and again placed in a muffle for 20–30 min. The procedure is repeated until the dry residue turns yellowish and the particles of black coal cease to appear. Then, 5 ml of hydrochloric acid was added to the cooled sample in a 1/1 ratio. The solution was transferred to a 25 ml flask and filled with distilled water. In the same way, a null solution was prepared using an empty amount. The samples were analyzed using an Atomic Absorption Spectrophotometer—Perkin Elmer A Analyst 200.
Addition of EDTA to determine the effect of the ability of Spirulina on the absorption of heavy metals
Because of the chelating properties of EDTA, it has been useful to figure out how to increase the absorption capacity of Arthrospira platensis about heavy metals. Thus, laboratory analyzes were performed on Spirulina plus EDTA to compare the number of heavy metals penetrate the Spirulina mass over the next 120 h.
Spirulina biomass was 10 mg L−1. The incubation was carried out in a 50 × 20 × 25 glass aquarium (in cm, length × width × height) with constant air barbotage (air flow rate 2 L min−1), temperature 25 °C, with the following lighting conditions: 24 L/0 D, PPFD 15 μmol·m−2·s−1. These experiments were carried out on both heavy metals, copper and lead to be investigated. A sample of Arthrospira platensis was taken from the incubation area for test in 1-L flasks. There were added salts CuSO4 and Pb (NO3)2, respectively, for artificial pollution with copper and lead (100 ppm). Two control samples were taken, to check the metal content in them. Control a0 (Arthrospira platensis), and Sample b0 (Spirulina platensis + EDTA). In the other samples, the chelating agent was added twice than copper and lead accordingly. Sampling was carried out for 4 h, 1 time during the first day and the following days, 1 time per day, including the fifth day 20 ml. Sample was centrifuged for 5 min at 1000 g to get 2 g of Spirulina biomass. Contain lead and copper were determined by the atomic absorption method in the got mass of Spirulina.
The influence of biosurfactants on the absorption of heavy metals (Cu2+and Pb2+) by Spirulina
Microbial synthesis of the Rhamnolipid biosurfactants was conducted using the Pseudomonas sp. PS-17 strain
(from the collection of Department of Physical Chemistry of Fossil Fuels of InPOCCC, National Academy of Sciences of Ukraine). The strain synthesizes homologous extracellular Rhamnolipids and extracellular biopolymer, which form a surface-active complex with Rhamnolipids (Semeniuk et al. 2020).
The Rhamnolipids, RL1, and RL2, contain one and two Rhamnose residues respectively, and two residues of 1-β-hydroxidecanoic acids. Containing RLs and TRL were determined spectrophotometrically (UVmini–1240, Shimadzu, Japan) using the orcinol method. The Rham- nolipids were isolated by extraction with Folch mixture (chloroform–methanol 2:1) which was further separated and analyzed using thin-layer chromatography (Lubenets et al. 2013).
For finding out the effect of biosurfactants, two different Rhamnolipids were used: Rhamnolipid1 (mono-Rhamnolipid) and Rhamnolipid2 (di-Rhamnolipid), also Trehalose lipid. Spirulina biomass was sampled in the one-liter flask to analyze each biosurfactant and heavy metals. For artificial pollution with copper and lead, lead nitrate and copper sulfate salts were used, respectively. Analysis was carried out for three kinds of biosurfactants in different flasks. The number of biosurfactants was added to 0.01% of the total solution.
The combination of analytical samples was distributed: Spirulina+copper+RL1; Spirulina+copper+RL2; Spirulina + copper + TRL; Spirulina + lead + RL1; Spirulina + lead + RL2; Spiruluna + lead + TRL. Analytical samples were taken 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h after application of the biosurfactants. The analyses were performed by the atomic absorption method. The computation of results was carried out in Microsoft Excel (Version 2019). The statistics were subjected to a one-way analysis of variance. The data presented represent the mean of triplicates ± standard deviation. Statistical analysis was done by ANOVA.
Results and Discussion
Results of different concentration Cu2+and Pb2+ ions on the formation of biomass and chlorophyll of Spirulina
As a result, Spirulina will exhibit its remedial properties in 100 ppm polluted water. As observed on Fig. 1. Copper and Lead cannot inhibit the main physiological indicator (biomass) of the developed Arthrospira platensis.
The results shown in Fig. 2 illustrate heavy metals are not an obstacle to the biological production of chlorophyll. In 100 ppm copper solution, chlorophyll formation is suppressed by only 25%. As a result, the process of photosynthesis lasts since heavy metals (copper and lead) do not destroy it. Absorb solar energy and its transformation into the chemical energy of organic substances continue. As observed, there are no significant changes that create chlorophyll.
The number of metals absorbed by the algae was determined by atomic absorption spectroscopy. As shown in diagrams, in the control samples, the heavy metals existing in the pure Spirulina biomass were equal to 0. As a result, lead penetration is 3.5 times higher than copper. Figure 3 shows data for both metals. Maximum absorb was observed at 72 h for 5 days.
Effect of EDTA on the absorption of heavy metals (Cu2+and Pb2+) by Spirulina during the 120 h
Since adding EDTA, the results show that the ability to absorb Spirulina is increased in the first 8 h for both metals. However, the maximum amount of absorbed metal differs between copper and lead samples. From a copper-contaminated environment, it boosted by 63%. In contrast, it decreased with lead by 10%. Thus, EDTA only helped speed up the penetration process (Figs. 4 and 5).
It can be assumed that chelating copper with EDTA is more easily absorbed by Spirulina than lead. Probably, the fact that lead is slightly more than 3 times heavier than copper plays an important role in this respect. It was interesting for us whether the same trend would show up in the difference between copper and lead in the case of biosurfactants.
Influence of biosurfactants on the ability of Spirulina to purify water. Results of absorption of Cu2+and Pb2+ ions with the combined action of Spirulina and biosurfactants
According to the results, the lipid Rhamnolipid1, Rhamnolipid2, and Trehalose showed unique properties in combination with Spirulina for both metals (Figs. 6 and 7). As shown in the comparable graphs, RL1 accelerated the absorption process during the first 8 h. RL2 shifted the absorption peak from 72 to 48 h for lead contamination. But with copper, the peak remains for 72 h. However, the maximum amount of absorbed metals was increased by copper, not lead. The Trehalose lipid showed better results than both rhamnolipids in absorbing heavy metals by Spirulina platensis, which will be taken into account in process of creating a wastewater treatment technology. Biosurfactants activate the remedial abilities of Spirulina—particularly, RL1 increased the maximum amount of absorbed Cu2+ ions by 42%, RL2 increased the maximum amount of absorbed Cu2+ ions by 68%, and TRL by 73%. The action of biosurfactants is even more important because of their environmental friendliness.
In bioremediation, scientists have studied different methods to improve the remediation skills of Spirulina. For that reason, some add carbohydrates to increase the number of absorbed metals (Markou et al. 2015). Others studied the intake capacity of dry and raw Spirulina and discovered that dry was more skillful at purifying water than raw one (Al-Homaidan et al. 2014). However, the increase picked up in both investigations does not exceed 5%. Still, we involved improving the absorption of heavy metals by algae in less time. And the amount of absorbed copper increased by 73% with Trehalose lipid.
Here, should be noted—after reaching the maximum concentration of metal in Spirulina biomass—at 72 h, the metal returns to the incubation medium. This requires further research. At this stage, one can only assume that this may be because of the osmotic gradient. Of course, this behavior of Arthrospira platensis must be considered in remediation methods against metals. The release of metal from Spirulina into the solution after 72 h of incubation occurs less in experiments with the EDTA.
The advantages of Spirulina are that it can multiply in salty and even alkaline environments, which are a barrier to other microorganisms. Still, many studies prove the ability of various microorganisms to absorb heavy metals and their apparent ability to purify water. However, Spirulina is the cheapest, healthiest and friendliest product, as its medicinal properties were researched many centuries ago.
Conclusions
This paper clarifies the role of biosurfactants in enhancing the remediation abilities of Spirulina in copper contaminated water. The presented study showed the ecological potential of Arthrospira platensis concerning heavy metals. The data got revealed their joint action. A series of tests show that absorb Cu2+ ions into Spirulina biomass was improved and sped up by the use of chelating agents and producents of bacteria—biosurfactants. EDTA increased the absorption of copper ions by Spirulina by 63%, Rhamnolipid1 by 42%, Rhamnolipid2 by 68%, and Trehalose lipid by 73%. However, Spirulina itself absorbs 29% more lead from a lead-contaminated environment than by consortium with Trehalose lipid. So, from the lead-contaminated water, the chelating agents only sped up the absorption process. Elimination of copper and lead ions from the contaminated site within 72 h was shown to speed up this process to 48 h after additional biosurfactants.
This topic can provide a cheap and friendly approach to purifying wastewater from lead and copper ions. Further studies will include an ultrastructural analysis of Spirulina and reveal the mechanisms of sorption and desorption of metals by Spirulina more accurately also Mass-spectrometry provides important information that leads to the identification and quantification of the individual capabilities of bio-surfactants—how they can improve the penetration of heavy metals into the Spirulina platensis. As a result, the basic idea of mechanisms for improving and accelerating the absorption properties of heavy metals by biomass Arthrospira platensis with the addition of ecological additives-biosurfactants was implemented with a reasonable result.
Acknowledgements We would like to thank our colleagues for their backing and support: Professor Dr. Dr. h.c. mult. Angelika Ploeger, Sustainable Agriculture and Food Systems (SAFS), Project Director – VW, Kassel University, Germany; Professor Dr. Dr. h.c. mult. Hartmut Vogtmann – Kassel University, Germany; Professor Vakhtang Lezhava Rector of the Agricultural University of Georgia; Professor Dr. Teo Urushadze, Dean of Agricultural and Nature Science School at the Agricultural University of Georgia, SAFS program director from Georgia; Natia Samushia Vice-Rector of the Agricultural University of Georgia; Professor Nato Kobakhidze, Ph.D. School coordinator of the Agricultural University of Georgia; Nino Katcharava SAFS program coordinator.
Author contributions All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by [IT]; Conceptualization: [IT] and [TV]; Methodology: [GK], [MK] and [MP]; Formal analysis and investigation: [IT] and [LC]; The first draft of the manuscript was written by [IT] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript; Writing – review and editing: [PvF und N], [OK] and [VL]; Resources: [OK] and [VL]; Supervision: [TV] and [Pvon F und N].
Funding This work was supported by co-financing (No 04/47) of Shota Rustaveli National Science Foundation (SRNSF) and Volkswagen Foundation, the Doctoral Program “Sustainable and Agricultural Food Systems” (SAFS).
Availability of data and material The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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