Journal of Microbial & Biochemical Technology

Spirulina platensis and Chlorella vulgaris Assisted Bioremediation of Heavy Metal Contaminated Aquatic Ecosystem

Avinash R. Nichat^1*, S. A. Shaffi² and V. K. Kakaria^{2 1}Department of Zoology, Government P.G. College Bhakhara, Dhamtari, India
²Regional Education of Institute (NCERT), Bhopal-13, India
^*Correspondence: Avinash R. Nichat, Department of Zoology, Government P.G. College Bhakhara, Dhamtari, India, Email:

Received: 28-Mar-2020 Published: 31-Jul-2020

Introduction

Among the pollutants heavy metals are regarded as one of the most serious pollutants are due to their environmental persistence and tendency to concentrate in aquatic organisms. Heavy metals are chemical elements with a specific gravity that is at least five times greater than specific gravity of water and the pollution of ecosystem by heavy metal is an important problem. Heavy metals constitute some of the most hazardous substances that can bio-accumulates [1-4].

Heavy metals further affect organisms directly by accumulating in their body or indirectly by transferring to the next tropic levels of the food chain [5-7]. The accumulation of heavy metals in the viscera, precipitation leads into chronic illnesses and cause significant damage to various organisms including induced stress, lipid per oxidation, protein denaturation, DNA damage, decreases organism’s life span and productivity of the natural water body [8,9].

The physiological, cellular & molecular mechanisms too used to regulate & detoxify environmental heavy metal toxicity on a variety of organisms but a clear understanding about the mechanism is awaited and expect further studies to establish a clear understanding on the above matter & through food & water, heavy metals/pollutants invariably find a place in the organisms including humans [2,6].

Heavy metal induces oxidative damage in different organs by increasing per-oxidation of membrane chemistry and altering the antioxidant system of the cells/tissues [10,11]. Interaction of metal ions with the cell organelles cause injury to cellular components. Heavy metal intoxication further depletes glutathione & protein bound sulfahydryl groups resulting into the production of reactive oxygen species like hydrogen peroxides, superoxide ions & hydroxyl radicals. These reactive oxygen species induce elevated visceral per-oxidation [9,12,13].

Researchers are innovating novel methods to clean up the heavy metal polluted water bodies by replicate transitional physical & chemical methods of environmental cleanup through phytoremediation [10,14,15].

Hence the need of the man is to innovate some alternative technologies &devices to protect the nature gifted consumables and to boost the yield from natural water bodies [8,12,16]. Based on the above information, the autotrophic microbes like used as detoxifying agent on few economically, nutritionally & culturally important fish species have been selected for the present study.

The aim of this investigation is to determine the safety, sub-lethal and lethal concentration of copper & zinc, their impact on the bio-chemical compartmentation of carbohydrate metabolism enzymes phosphoglucomutase, hexokinase, phosphoglucoisomerase and phosphofructokinase in different brain regions of three nutritionally & economically important fish species i.e, Labeo rohita (Ham.), Clarias batrachus (Linn.) and Channa punctatus (Bloch.) on a comparative basis.

Material and Methods

Alive, healthy, mature, disease-free & active Labeo rohita (Ham.), Clarias batrachus (Linn.) and Channa punctatus (Bloch.) 120-130 gm of 18-20 cm (standard length) were obtained from few selected local ponds to avoid ecological variation and acclimatized in the laboratory condition for a period of seven days and were subjected for various exposures and investigations.

Determination of safety, Sub-lethal and lethal concentration

Safety, sub-lethal concentrations of copper was determined on Labeo rohita, Clarias batrachus and Channa punctatus by the Probit Analysis Method [17]. Higher concentration of copper was used and slowly reduced the amount of concentration to know the Lc 50/100 value for 96-hour exposure.

Acute studies

The Labeo rohita, Clarias batrachus and Channa punctatus (120-130 gm) of 18-20 cm (standard length) were taken separately and kept in twenty groups and each group consist of forty-eight fish species. No food was given to the above fish species during this period (08, 16 & 24hrs). The first set of Labeo rohita, Clarias batrachus and Channa punctatus were exposed to sub-lethal and lethal concentration of copper and zinc the detail were described somewhere else [14].

Preparation of tissue extract

The termination of the experiment preparation of tissue extract and enzyme assays were described elsewhere [18,19].

Statistical analysis

The experiments with acute and chronic studies were repeated at least seven times separately to subject the data for analysis of variance.

Results

The results enlightened that the combined influence of both the microbes (Chlorella vulgaris & Spirulina platensis) heavily decreased the toxic influence of copper & zinc on carbohydrate enzymes (phosphoglucomutase, hexokinase, phosphoglucoisomerase & Phosphofructokinase) in brain regions (cerebrum, diencephalons, cerebellum & medulla oblongata) in Labeo rohita (sub-lethal concentration of Zn- 0.72 mg/ltr, Cu- 0.10 mg/ltr), Clarias batrachus (sub-lethal concentration of Zn- 2.75mg/ltr, Cu- 0.50 mg/ltr), and Channa punctatus (sub-lethal concentration of Zn- 2.90mg/ltr, Cu- 0.80mg/ltr) under chronic studies (Table 1-8).

The sub-lethal copper concentration (in presence of two microbes) inhibited the phosphoglucomutase to a significant extent at thirty days exposure than in cerebrum, medulla oblongata & cerebellum in comparison to 15- & 45-days exposure in Labeo rohita (Table 1). In Clarias batrachus the fall in phosphoglucomutase was maximum in diencephalons at 30-day exposure followed by cerebrum, medulla oblongata at 15 days exposure & cerebellum at 30 days exposure than at 45 days exposure (Table 1).

Table 1: Summarize of 16 Non-structural Proteins (NSP) in coronavirus and their function

In Channa punctatus the fall in phosphoglucomutase was highest in diencephalons at 30 days of exposure than in cerebrum, medulla oblongata & cerebellum at 15 days of exposure than at 45 days of exposure under chronic studies (Table 1).

The combined influence of Chlorella vulgaris & Spirulina platensis was experimented on sub-lethal concentrations of copper toxicity in which hexokinase registered optimum fall in diencephalons at 30 days of exposure followed by cerebrum & medulla oblongata at 15 days of exposure & cerebellum at 30 days of exposure than at 45 days of exposure in Labeo rohita (Table 2). In Clarias batrachus the hexokinase fall was recorded in diencephalons to a great extent at 15 days of exposure than in cerebrum, medulla oblongata & cerebellum in comparison to 30- & 45-days exposure (Table 2).

Table 2: Combined influence of Chlorella vulgaris & Spirulina platensis on copper metal (sub-lethal) caused toxicity in three freshwater teleosts Hexokinase Chronic studies

The hexokinase maximum fall was at 30 days exposure in diencephalons followed by cerebrum, medulla oblongata and cerebellum at 15 days of exposure than 45 days exposure under chronic studies in Channa punctatus (Table 2).

The phosphoglucoisomerase fall was optimum in diencephalon accompanied by cerebrum, medulla oblongata & cerebellum at 15 days exposure in Labeo rohita (Table 3) exposed to sub-lethal concentrations of copper in microbe’s presence. In Clarias batrachus (Table 3) the phosphoglucoisomerase fall was highest in diencephalons at 30 days exposure to sub-lethal concentrations of copper in comparison to cerebrum, medulla oblongata & cerebellum at 15 days of exposure. The fall in phosphoglucoisomerase was noticed in diencephalons at 15 days of exposure accompanied by cerebrum, medulla oblongata & cerebellum under chronic studies in Channa punctatus (Table 3) than at 30 & 45 days of exposure.

Table 3: Combined influence of Chlorella vulgaris & Spirulina platensis on copper metal (sub-lethal) caused toxicity in three freshwater teleosts - Phosphoglucoisomerase - Chronic studies

The fall in phosphofructokinase was maximum in diencephalons at 30 days of exposure to sub-lethal concentrations of copper in presence of two microbes (Chlorella vulgaris & Spirulina platensis) in comparison to cerebrum, medulla oblongata (15 days exposure) & cerebellum (30 days exposure) in Labeo rohita (Table 4). In Clarias batrachus (Table 4) the fall in phosphofructokinase was noticed in diencephalons than in cerebrum, medulla oblongata & cerebellum at 15 days of exposure. The fall in phosphofructokinase was optimum at 30 days in dienecephalon accompanied by cerebrum, medulla oblongata and cerebellum at 15 days exposure to sub-lethal levels of copper in the microbe’s presence in Channa punctatus (Table 4).

Table 4: Combined influence of Chlorella vulgaris & Spirulina platensis on copper metal (sub-lethal) caused toxicity in three freshwater teleosts - Phosphofructokinase - Chronic studies

At 30 days exposure to sub-lethal concentrations of zinc the presence of two microbes affected phosphoglucomutase at 30 days in diencephalons than in cerebrum, medulla oblongata at 15 days & cerebellum in the Labeo rohita (30 days exposure) (Table 5). In Clarias batrachus (Table 5) the variations recorded in the phosphoglucomutase was prominent in diencephalons at 30 days followed by cerebrum, medulla oblongata (15 days exposure) and cerebellum (30 days exposure) than at 45 days exposure. The phosphoglucomutase fall was significant at 30 days in diencephalons in comparison to cerebrum, medulla oblongata (15 days exposure) & cerebellum under long term studies in Channa punctatus (Table 5).

Table 5: Combined influence of Chlorella vulgaris & Spirulina platensis on zinc metal (sub-lethal) caused toxicity in three freshwater teleosts - Phosphoglocomutase - Chronic studies

The maximum fall in hexokinase in the presence of two microbes exposed to sub-lethal concentrations of zinc was in diencephalons at 30 days than cerebrum, medulla oblongata & cerebellum (15 days exposure) in Labeo rohita (Table 6) in comparison at 45 days of exposure. In Clarias batrachus (Table 6) the fall in hexokinase was noticed in diencephalons at 30 days prominently in comparison to cerebrum, medulla oblongata & cerebellum than at 15- & 45-days exposure. In Channa punctatus (Table 6) to the fall in hexokinase was significant in diencephalons at 30 days exposure accompanied by cerebrum, medulla oblongata (15 days exposure) and cerebellum (30 days exposure) under long term studies.

Table 6: Combined influence of Chlorella vulgaris & Spirulina platensis on zinc metal (sub-lethal)caused toxicity in three freshwater teleosts - Hexokinase - Chronic studies

The fall in phosphoglucoisomerase was maximum at 30 days in diencephalons in comparison to cerebrum, medulla oblongata (15 days) & cerebellum (30 days) exposed to sub-lethal concentrations of zinc in the presence of two mibrobes in Labeo rohita (Table 7) than at 45 days of exposure. In Clarias batrachus (Table 7) the phosphoglucoisomerase fall was highest in the diencephalons at 30 days of exposure to zinc in the presence of microbes than 15- & 45-days of exposure than in cerebrum, medulla oblongata & cerebellum (15 days exposure). The sub-lethal concentrations of zinc manifested optimum enzyme variation in diencephalons at 30 days than at 15- & 45-days exposure accompanied by cerebrum, medulla oblongata (15 days exposure) and cerebellum (30 days exposure) under long term studies in Channa punctatus (Table 7).

Table 7: Combined influence of Chlorella vulgaris & Spirulina platensis on zinc metal (sub-lethal) caused toxicity in three freshwater teleosts - Phosphoglucoisomerase - Chronic studies

The sub-lethal concentrations of zinc in presence of two microbes described earlier manipulated phosphofructokinase (Table 8) to a marked extent in diencephalons at 30 days exposure than in cerebrum, medulla oblongata (15 days exposure) and cerebellum (30 days exposure) in Labeo rohita. In Clarias batrachus (Table 8) also the diencephalon phosphofructokinase registered highest fall at 30 days exposure than in cerebrum, medulla oblongata (15 days) & cerebellum (30 days) exposure to sub-lethal level of zinc in presence of microbes.

The trend in phosphofructokinase fall exposure to sub-lethal concentrations of zinc in the presence of two microbes in Channa punctatus is more of less similar to Labeo rohita & Clarias batrachus under chronic studies (Table 8).

Table 8: Combined influence of Chlorella vulgaris & Spirulina platensis on zinc metal (sub-lethal) caused toxicity in three freshwater teleosts - Phosphofructokinase - Chronic studies

Discussion and Conclusion

The mechanism of detoxification of copper & zinc may be visualized as aquatic autotrophs has been used in industrial but also in domestic uses like water treatment as they are capable of removing waste to a great extent.

The uptake of copper & zinc by aquatic autotrophs used in present investigation has been realized that the aquatic autotrophs have an initial rapid stage and a slower stage. During rapid phase the metal ions are absorbed on the surface and transport them across the cell membrane & it is the first symptom of cell damage & deterioration of membranes [4,10,12,20].

It is further observed that there is an increase in the number of polyphosphate bodies with heavy metal toxicity in cyanobacteria as polyphosphate bodies have been working as indicators of metal absorption in cyanobacteria. The strong negative surface charge of polyphosphate in the phosphate bodies may help in absorbing the metal. The polyphosphate bodies may contain magnesium, sodium, iron & phosphorous. The polyphosphate bodies may not function in storage of polyphosphate but also help in detoxification mechanism [10,14,21,22].

The cyanobacteria further contain cyanophin granules that act as storage in the cell. Perhaps these bodies may participate in the cell internal detoxification process. The pH of the media may also influence the toxicity of copper & zinc by altering the form/nature of heavy metals [2,20].

Hydrogen ions may also play a vital role to check the toxic impact of copper & zinc as the metal binding sight on the cell surface binds with a proton reflects. Those protons will compete with metal ions for the binding sight. Change in gases ratio in aquatic system may change the temperature of the media by that aquatic autotrophs may absorb heavy metals [23-25].

The above-mentioned episodes are not totally/partially ruling out even in the present investigation and the fall in phosphoglucomutase, hexokinase. phosphoglucoisomerase & phosphofructokinase in cerebrum, diencephalons & medulla oblongata in Labeo rohita, Clarias batrachus & Channa punctatus with direct sub-lethal and lethal metal exposure and in presence of aquatic autotrophs & their cell organization bound mechanism of' detoxification to neutralize the sub-lethal & lethal copper & zinc concentrations affect the acute & chronic studies prominently reflect that without the possibilities of the above mentioned & discussed processes it was not possible for Chlorella vulgaris & Spirulina platensis to detoxify the metal caused toxicity on phosphoglucomutase, hexokinase, phosphoglucoisomerase & phosphofructokinase in Labeo rohita, Clarias batrachus & Channa punctatus on a comparative basis from a tropical habitat.

The finding may help to understand the microbe-metal interaction and sub sequent detoxification of the metal to a less extent in a better way. The sub-cellular regions of Cyanobacteria and Anabaena cylindrica could trap the lead through its phosphate and precipitates in the form of lead phosphate on the cell wall inside the cell [8,9,12,26-28].

The following mechanisms are used for microbial bioremediation:

(1) Sequestration of toxic metals by cell wall components or by intracellular metal binding proteins and peptides such as metallothioneins (MT) and phytochelatins along with compounds such as bacterial siderophores which are mostly catecholates, compared to fungi that produce hydroxamate siderophores.

(2) Alteration of biochemical pathways to block metal uptake.

(3) Conversion of metals to innocuous forms by enzymes.

(4) Reduction of intracellular concentration of metals using precise efflux systems.

The mechanisms used in remediation of heavy metals from contaminated soils are presented in following (Figure 1).

Figure 1: The mechanisms used in remediation of heavy metals from contaminated soils are represented.

Figure 2: Mechanisms of biosorption based on (a) Dependence on cell metabolism; (b) Location within the cell where the metal is removed.

Mechanisms of removal of heavy metals from contaminated soils by microorganism through the processes of precipitation, biosorption via sequestration by intracellular metal binding proteins (metallothioneins) and conversion of metals to innocuous forms by enzymes (enzymatic transformation).

Similar kind of mechanism might have taken place in the present findings i.e., less fall of enzymes in which the cellular components of Spirulina platensis might have precipitated the metal into compound with the help of its cellular components and the present findings i.e., less fall of enzymes in presence of a autotroph than the enzyme fall when directly exposed to copper & zinc sub-lethal & lethal levels should understand on similar lines. Enhanced polyphosphate bodies formation was ascribed to heavy metal toxicity exposed group of animals and perhaps these bodies were suggested as the site of metal absorption in aquatic autotrophs [13,20,27,29,30].

The physico-chemical factor of the water body is most affected due to continuous discharge or dumping toxicants from different source. Alteration in the physico-chemical parameters of the habitat would generate stress and this stress not only influences other organisms but their function including of the water. The pH of the water media certainly reduces the toxicity of cadmium & zinc to the fish in general and nervous system in particular and forms a compound with hydroxyl group such a mechanism may not be ruled out as Spirulina platensis has a higher absorption capacity for heavy metals and the fall in the above said enzymes is less in aquatic microbe presence than in direct metal exposure i.e., the metal complex formation with hydroxyl group might be higher in the diencephalons in comparison to cerebrum, medulla oblongata & cerebellum in Labeo rohita than in Clarias batrachus & Channa punctatus.

The heavy metal removal significantly affected by the pH in the solution as hydrogen ions plays an important role in multicomponent absorption system. The increase in heavy metal uptake by autotroph Spirulina platensis & Cynobacteria with the increasing pH. A pH dependence of ion generally occurs when heavy metal binding site on cell surface binds with proton. This indicate that the protons will compete with metal ions for the binding site. Hence most ions are absorbed at a highest pH in a better way due to lower competition with protons. This indicates that heavy metals were smartly absorbed in a pH range of 4-8 [11,23,31,32].

The potential negative surface charge of the poly-phosphate in the polyphosphate bodies will assist to absorb metal. Increase in the exposure time of autotrophs to heavy metals further increase the number of polyphosphate bodies & also composed of other materials such as magnesium, sodium, potassium, iron & copper [12,20,34-36]. Such bodies not only function in polyphosphate storage and further functions as a detoxification process such a mechanism is not rule out even in the present investigation and the fall of phosphoglucomutase, hexokinase, phosphoglucoisomerase and phosphofructokinase with the metal exposure directly on one side and metal exposure in presence of Spirulina in Labeo rohita, Clarias batrachus & Channa punctatus on both side educates that the presence of the aquatic autotroph significantly checked the fall off the enzymes in different brain regions of the above said fish species is quite innovative and need further investigation on a large scale for the application in the aquatic system and to check the menace of pollution [3,12,15,37].

This investigation further helps that aquatic autotrophs can be used to remove heavy metals from aquatic system over a wide range of pH. Such events might have taken place even in the present investigation and the less fall in phosphoglucomutase, hexokinase, phosphoglucoisomerase & phosphofructokinase in different brain regions of Labeo rohita, Clarias batrachus & Channa punctatus might be ascribed to a less degree in microbe presence than direct exposure to heavy metals.

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