Data obtained from points CM and P14 regarding biological variables
The analysis of the phytoplankton community, represented by the microalgae and cyanobacterial community, was performed by grouping the individuals by taxonomic classes, and the results obtained for points CM and P14 are represented in Fig. 2A and 2B, respectively.
The microalgae and cyanobacteria community found in CM was represented by 25 taxa divided into 7 classes, while for P14 it was represented by 28 taxa divided into 8 classes. Although the point CM correspond to the effluent in natura, being under the direct effects of AMD, a taxonomically diverse microalgae flora was observed, being dominated by the classes Bacillariophyceae, corresponding to 32% and Chlorophyceae with 24% of the taxa, which together represent more than half of all classes found for this point, as can be seen in Fig. 2A. For P14, the classes that most contributed to the total composition of the phytoplankton community at this point of the Antas Reservoir in the analyzed periods were the same predominant in the point CM, differing only in percentage, being Chlorophyceae with 32% and Bacillariophyceae with 21%, as shown in Fig. 2B.
Regarding the individual density of each genus and/or species registered within each class, this data was not obtained. The total density data by taxonomic class were obtained from Azevedo et al. (2010) and are shown in Table 1.
Table 1
Density values of individuals belonging to the microalgae and cyanobacteria community represented by taxonomic classes in water samples from CM and P14
| | | CYA | CHL | EUG | BAC | ZYG | CHRY | DIN | CRYP |
| Out/08 | CM | 0 | 0 | 0 | 220 | 2 | 0 | 0 | 2 |
| P14 | 5 | 8 | 50 | 34 | 10 | 10 | 33 | 23 |
| Jan/09 | CM | 250 | 25 | 57 | 33 | 1 | 0 | 0 | 8 |
| P14 | 107 | 96 | 124 | 242 | 0 | 0 | 34 | 11 |
| Apr/09 | CM | 0 | 13 | 57 | 86 | 0 | 0 | 6 | 115 |
| P14 | 30 | 0 | 30 | 0 | 0 | 0 | 932 | 30 |
| Jul/09 | CM | 0 | 205 | 0 | 4 | 0 | 0 | 0 | 4 |
| P14 | 2 | 5 | 74 | 41 | 7 | 9 | 18 | 2 |
| TOTAL | CM | 250 | 243 | 123 | 343 | 3 | 0 | 6 | 129 |
| P14 | 144 | 109 | 278 | 317 | 17 | 19 | 1017 | 16 |
| Unit: Ind.ml-1. CYA: Cyanophyceae; CHL: Chlorophyceae; EUG: Euglenophyceae; BAC: Bacillariophyceae; ZYG: Zygnematophyceae; CHRY: Chrysophyceae; DIN: Dinophyceae; CRYP: Cryptophyceae |
For CM, the Bacillariophyceae class, encompassed the highest percentage of taxa recorded, and also presented the highest value of total density of individuals, corresponding to 343 ind.mL-1. The Cyanophyceae class, although it appeared only in January/09, presented a high density of individuals, with 250 ind.mL-1, followed by the Chlorophyceae class with 243 ind.mL-1.
It is striking that in July/09 only one taxon of each class was identified, and of these, Botryococcus sp stood out, presenting a high density of 205 ind.mL− 1. As in the studied region, July is a dry month, the lack of rain may have left the metals less diluted, and consequently affected the composition of the local phytoplankton. The fact that Botryococcus has excelled is due to its ability to survive in acidic conditions and with high concentrations of metals, even if its photosynthetic and respiration rates are affected (Areco, Haug, Curutchet, 2018).
On the other hand, for P14 the Chlorophyceae class was dominant in relation to the percentage of representatives, but the Dinophyceae class stood out in relation to the density of individuals, with a value of 1017 ind.mL− 1, followed by Bacillariophyceae with 317 ind.mL− 1 and Chlorophyceae with 278 ind.mL− 1. The class with the lowest total density of individuals was Zygnematophyceae with only 17 ind.mL− 1.
The results presented in this study showed that, in the untreated effluent samples from the mine pit at INB, affected by AMD, with acid pH and high concentrations of stable and radioactive contaminants (Ferrari et al. 2015) there is a microalgae biodiversity consisting of 25 representatives, a relatively high number when compared to that found by other authors, such as Baselga-Cervera et al. (2020) and Kumar et al. (2016) who found only 5 taxa in their studies in acid lakes mining. This may be related to pH, which is not extremely acid in the effluents of the pit mine of the INB, being around 3.8 (Ferrari et al., 2017). In the acid lakes of Lusatia, the number of taxa is around 7 and 9 when the pH is between 2.5–3, and when the pH is around 4, the number of taxa goes to 17–25 (Geller et al., 2013).
In the same way that in the present study, Gomes et al., (2021) also found a diverse microalgae community when analyzing AMD samples from the São Domingos mine in Portugal, corresponding to 14 taxa divided into 8 classes, dominated by Bacillariophyceae, as found in the present study.
The CM point is a point that suffers directly from AMD and is characterized by its acidity and high concentrations of metals such as uranium, zinc, aluminum and manganese (Ferrari et al., 2017). Even so, several representatives of different taxonomic classes were found in the CM point, revealing an excellent ability of these organisms to survive in adverse conditions.
Prasanna et al. (2011), when analyzing the microalgae community in waters under the effects of AMD, with pH between 2.4–3.2, found a high diversity, with representatives of the genera Euglena, Cylindrocystis, Botryococcus and Navicula, as found in the present study. These authors suggest that such a high diversity of microalgae may be related to the long duration of AMD flow, leading to adaptation of these organisms (Prasanna et al., 2011). Therefore, this is an explanation that can also apply to the present study.
When a comparison is made between CM and P14, it is possible to observe that many microalgae were common to both sites, such as Microcystis sp, Dictyosphaerium pulchellum, Trachelomonas sp, Euglena sp, Eunotia sp, Navicula spp, Peridinium sp, Cryptomonas sp, among others.
The adaptation ability of these organisms, since they are present in both locations, may be related to the appearance of resistant cells spontaneously emerged in populations in equilibrium (Baselga-Cervera et al., 2020).
Another factor that may justify the appearance of these organisms in both sites would be the presence of biological mechanisms to support a wide range of pH and high concentrations of metals. This is the case of Euglena, a genus commonly reported in natural or anthropogenic acid waters rich in metals, as the cell can limit the access of toxic elements to their interior through the process of adsorption of metals to the cell wall, thus enabling their survival under stress conditions (Valente, Gomes, 2007).
Regarding the P14 sampling point, representatives of the Chrysophyceae class were found, which is an absent class in CM. In general, according to Wehr, Sheath and Kociolek (2015), this class is associated with waters with pH between 6 and 7, which justifies the fact that it was not found in CM.
The prominent class in P14 is Dinophyceae, with the Peridinium sp, which presented high total density throughout the study period, as previously shown. Peridinium can also be found in acid waters (Lessmann, Fyson, Nixdorf, 2000; Kalin et.al., 2006) and was present in CM, but at much lower densities than in the Antas Reservoir. When analyzing water samples from the Antas Reservoir, Ronqui (2008) also highlighted the dominance of Peridinium sp with regard biomass and density, and numbers were even higher when analyzed at the point where there was better water quality. Probably, this preference of Peridinium sp for sites with better water quality is the reason why it occur in higher numbers in P14 than in CM.
Biotechnological Potential Of Phytoplankton In Extreme Environments
Chlamydomonas sp, Desmodesmus sp and Botryococcus sp, which play important roles related to biotechnology and environmental remediation, were found in CM, as well as Euglena sp, which was also found in P14.
Dean et al. (2019) studied the adaptation mechanisms of a strain of Chlamydomonas acidophila, and found that this organism was able to grow in a wide pH range, having its optimal growth range at pH 3–5. In addition, this strain, was able to grow in high cadmium concentrations, copper and zinc, with bioaccumulation inside the cell, showing tolerance to these metals due to metabolic adaptation, indicating that it may be useful in bioremediation.
Chlamydomonas sp also stands out with regarding to the remediation of environments contaminated with uranium. García-Balboa et al. (2013) studied the adaptation of microalgae in extremely polluted wastewater sites from an uranium mining area and found chlorophyta species, mainly Chlamydomonas sp, as being able to adapt quickly to these mine wastewaters. This was possible thanks to the genetic recombination achieved by sexual reproduction, in which resistant cells were the result of rare spontaneous mutations.
In addition, C. reinhardtii has been used as a model organism for studies aiming at the production of commercially viable biohydrogen (Iqbal et al., 2022; King et al., 2022).
Desmodesmus sp also plays important roles in bioremediation. Abinandan et al. (2020) carried out the first study that showed the potential of acid-tolerant microalgae, such as Desmodesmus sp, in iron removal and in biodiesel production when cultivated in extreme conditions.
Another interesting and very important application of Desmodesmus sp is in the oxidation of manganese. Wang et al. (2017), studying the application of Desmodesmus sp, found that this microalga has the ability to oxidize Mn (II), forming biogenic manganese oxides that have the potential to remove bisphenol A (BPA) from the environment. Such, biogenic manganese oxides generated by algae can be useful in the degradation of organic pollutants contained in water or sediment, thus contributing to environmental remediation.
Another microalga with excellent metal removal capabilities is Botryococcus sp. The authors Areco, Haug and Curutchet (2018) saw that Botryococcus braunii can remove zinc from aqueous solutions for long periods of time through adsorption processes and mainly by precipitation, thanks to its outstanding ability to reverse acidic conditions of the medium. Thus, this microalga is a strong candidate for bioremediation, because besides zinc, it can remedy heavy metals such as nickel and copper, and is an excellent option for use in environmental remediation measures in the aquatic bodies around the INB.
B. braunii is also considered an alternative source of biodiesel, because it can store high amounts of lipids, generating large amounts of oil (Maciel et al., 2020).
Euglena is another genus frequently reported in extreme environments and with several applications in biotechnology. For instance, Euglena gracilis, has many biotechnological applications, including: production of cosmeceutics and nutraceutics, nutritional supplement in aquaculture and animal feed, biomaterials, bioremediation of heavy metals, wastewater and ecotoxicological risk assessment (Krajcovic, Vesteg, Schwartzbach, 2015). Besides the production of metabolites, it is also useful in the production of biofuels and biogas (Khatiwada, Sunna, Nevalainen, 2020) and is one of the rare organisms capable of simultaneously producing antioxidants together with wax esters, phytotoxins and polyunsaturated fatty acids. Euglena lipids are rich in these polyunsaturated fatty acids, which have numerous medical applications: cancer prevention and treatment, cholesterol and blood pressure control, prevention of cardiovascular diseases, aid in brain development and are essential in child nutrition (Kottuparambil, Thankamony, Agusti, 2019).
Search For Patents
The search for patents, also known as technological prospecting, is important because it allows to identify the newest emerging technologies and how much they can be beneficial to society and the environment in general. In addition, it also allows for the expansion of knowledge in universities and of innovation in enterprises.
In this work, the search for patents was carried out in the WIPO database, as described in methodology. By placing the search term “microalgae” 3643 (three thousand six hundred and forty-three) patents were identified in the period 2017 to 2022. Of these 3643 deposited patents, the ones that most closely relate to the issues addressed in this work were analyzed, that is, patents on the use of microalgae viewing to bioremediation of contaminated areas. In this regard, the number of patents examined falls to 177, considering the same period, and the issues that have been addressed most in these patents are shown in Fig. 3.
As can be seen in Fig. 3, patents involving the use of microalgae for bioremediation of industrial wastewaters are predominant. The following are the applications for domestic wastewater treatment, water treatment and finally soils. In this approach, those patents concerning industrial processes were classified as industrial waste water related to mining, effluent treatment, aquaculture processes, oil refining, among others; as domestic waste water, those from sewage, baths, kitchens, among others; waters treatment included in situ remediation of rivers and lakes, water quality monitoring, bioremediation of water bodies, among others.
The main genera of microalgae used in the studies that originated these patents belong to Spirulina and Chlorella, none of which was recorded in the present study. In the patent registered by Qiang and Weixian (2019), the authors used the Chlorella W4 strain capable of removing heavy metals in water bodies, removing high rates of zinc, nickel and chromium, showing that its use is adequate for the removal of heavy metals in polluted waters.
Xiaodonh et al. (2021) also registered a patent with the use of Chlorella for removing heavy metals in water, this time using Chlorella vulgaris. The main goal of this patent was to provide charcoal-based particles of this microalga with high adsorption capacity, long service life and high efficiency of removal of heavy metals. As a result, they have seen that these charcoal-based microalgae particles display strong adsorption ability of ions of lead, copper, zinc, iron, cadmium, barium, aluminum, chrome and nickel.
Considering the total number of deposited patents involving microalgae, the number of patents related to the bioremediation area is still very low. Of the products originated from microalgae, those that stand out most regarding the number of patents are biofuels, followed by lipids and pigments. With regard to the application of products originating from microalgae, the main focus is health, energy and human nutrition (WIPO, 2016).
In general, the application of microalgae in bioremediation processes is still recent and this field needs more research and studies so that, in the not so far future, it will be possible to see such applications on industrial scale, rendering the processes more sustainable, thus contributing to the advance of green technology.