Identification of microalgae from waste stabilization ponds and evaluation of electroflotation by alternate current for simultaneous biomass separation and cell disruption

This work aimed to investigate algal diversity at the genus level in stabilization pond systems treating domestic wastewater and to evaluate the feasibility of an electroflotation by alternate current (EFAC) system for simultaneous microalgae separation and cell disruption. Evaluation of algal diversity showed that the genera Euglena and Chlorella were present in relatively high frequencies in five of the six effluents analyzed. The use of EFAC on an effluent that presented bloom of Chlorella achieved turbidity and chlorophyll-a removal efficiencies higher than 70 and 90%, respectively, after 70 minutes of operation. Total lipid yield for the Chlorella-rich biomass was 21.4±2.02%. Such high biomass lipid content demonstrates the potential for obtaining lipid-based biofuels from wastes. The current paper describes the first attempt, with promising results, at using electroflotation by alternate current for low cost, simultaneous microalgae harvesting and disruption.


INTRODUCTION
The use of non-renewable resources of fuels, agricultural nutrients and metals offers long-term sustainability issues for the current societal production system (LOVINS, 2008).The search for sustainable bio-based resources has been driving the production cycles in the world, in which raw material can be generated from technical and biological waste.
Wastewater treatment is a key platform for the technological development focused on the change of production system ( VAN LOOSDRECHT & BRDJANOVIC, 2014).Wastewater contains between 50 and 100% of lost waste resources.Therefore, the recovery and reuse of such resources Eng Sanit Ambient | v.24 n.1 | jan/fev 2019 | 177-186 Florentino, A.P. et al. have been the target of economists and environment experts (PUYOL et al., 2017).
Among the technologies contributing to the challenge of resource recovery from wastewater, biological ones, such as heterotrophic, chemotrophic, phototrophic, and photosynthetic bacteria, microalgae, and terrestrial plants, have been reaching highly efficient recovery of valuable resources from dilute streams (PUYOL et al., 2017;CHEN et al., 2017;RAZZAK et al., 2017).
Waste stabilization ponds are lentic environments built to treat wastewater by removing organic matter through biological processes (PEARSON, 2008).Microalgae play an important role in these ecosystems, consuming carbon dioxide and nutrients and producing oxygen that is used by bacteria to oxidize organic matter.Moreover, they can influence physicochemical conditions by modifying the color, turbidity, oxygenation, alkalinity and other properties of the ponds (RAWAT et al., 2011;BAHR et al., 2011;MUÑOZ & GUIEYSSE, 2006).
Although highly important during treatment, microalgae can have a negative impact on the quality of receiving water bodies due to their high content of organic matter, nitrogen and phosphorus, which can be used as substrate for bacterial growth (RAWAT et al., 2011;MULBRY et al., 2008;DE-BASHAN & BASHAN, 2010).
Unfortunately, most stabilization pond systems do not have a polishing step for microalgae removal before final discharge.If harvested during treatment in stabilization ponds, algal biomass can have commercial value due to its potentially high content of lipids, which could be used for the production of biofuels (RAWAT et al., 2011;DERNER et al., 2006).The lipid content of microalgae can vary considerably among species, ranging from 2 to 77% of their dry weight (CHISTI, 2007;MATA et al., 2010).
Despite the importance and great diversity of algae in waste stabilization ponds, there are few references in the current scientific literature to taxonomic surveys, and studies of the algal community structure of these environments (PHAM et al., 2014;PASTICH et al., 2016).Algal diversity in these systems is greatly influenced by the design parameters, environmental conditions such as temperature, solar radiation, domestic wastewater quality, etc., as well as operation conditions (PEARSON, 2008;MARA & HORAN, 2003).Thus, the diversity obtained in each system can influence the oil content that can be extracted from the biomass.
Biomass separation from water during production of biodiesel from algae may involve one or more steps and makes up to 20-30% of total production costs (RAWAT et al., 2011).The main reasons for high process costs are the small size of microalgae and their growth in very dilute cultures (biomass concentration lower than 1 g L -1 ) and similar density to water (BARROS et al., 2015;SHUBA & KIFLE, 2018).There is high demand for the development of appropriate and economically viable technologies for microalgal harvesting, which involves the combination of existing technologies or the development of brand new ones (MATA et al., 2010;GRIMA et al., 2003;BRENNAN & OWENDE, 2010;BARROS et al., 2015;RASLAVIČIUS et al., 2018;SHUBA & KIFLE, 2018).Current microalgal harvesting processes involve mechanical, chemical, biological and, to a lesser extent, electrical based methods.
Electrochemical processes generally generate hydroxyl radicals on the electrode (CHAPLIN, 2014;SIRÉS et al., 2014), and those radicals are responsible for the complete oxidation of the organic matter and its conversion to CO 2 and H 2 O.In this way, the undesirable compounds cannot be transferred between the phases of the process (PARSA et al., 2009).Therefore, the electroflotation process works as a solid/liquid separator based on the suspension of gas particles (hydrogen and oxygen) generated on the surface of the electrodes (ARAYA-FARIAS et al., 2008).In conventional flotation processes, the diameter of the gas bubbles produced varies from 600 to 1,000 µm.Due to the large size of the diameters, the gas bubbles cannot efficiently provide the flotation of particles with diameter smaller than 20 µm.However, bubbles generated by electroflotation have a diameter of 15 to 80 µm, which works efficiently in the flotation of small particles (SARKAR et al., 2011).Although the motion of an ion in solution is always uncertain, the presence of an electric field inserts a guided movement component and the ions migrate through the solution.In the frequency range applied, a resonance is obtained with the natural frequency of the water molecule, which breaks the atoms connections.This work aimed to investigate algal diversity at the genus level in stabilization ponds treating domestic wastewater and to evaluate the feasibility of electroflotation by alternate current (EFAC) for simultaneous separation and disruption of microalgal cells.

Sampling
Six waste stabilization ponds were selected for the evaluation of their phytoplankton diversity (Table 1).These systems were located in Fortaleza, Ceará, Brazil.Samples were collected near the outlet on the surface of the last pond at each system.For physicochemical analyses, samples were collected in 20 L plastic containers.
Samples for microscopic identification were collected using a plankton nylon net with 20 µm-pores (Sulpesca, Paraná, Brazil) and stored in sterile 200 mL glass containers.Although some microalgae have smaller sizes than the openings used, the filtered volume enables the collection of many species at random, allowing the study of phytoplankton diversity.

Physicochemical parameters
Samples were analyzed for pH, dissolved oxygen (DO), temperature, turbidity, chlorophyll-a, nitrite, nitrate, ammonia, total phosphorus, biochemical oxygen demand (BOD), chemical oxygen demand (COD) and alkalinity.All physicochemical parameters were determined according to Standard Methods for the Examination of Water and Wastewater (APHA;AWWA;WEF, 2008).

Microscopic identification
Samples were placed in a sterile glass container and fixed with Transeau solution (6 parts of water, 3 parts of 95% ethanol and 1 part of formaldehyde) in a 1:1 ratio (effluent:Transeau) (SILVA et al., 2014).Transeau solution was used only to preserve the morphology of microalgal specimens, and these samples were used exclusively in the microalgae identification assays.Slides were prepared with 20 µL of the prepared solution covered with a glass slide.
Identification of the dominant genus was carried out in five slidesand each slide was checked twice -with direct light in a trinocular optical microscope (L-1000T, Bioval, São Paulo, Brazil), using a 40X objective and taxonomic identification guides (BELLINGER & SIGEE, 2010;HUYNH & SEREDIAK, 2006).

Reactor design
The EFAC system used in this study to achieve simultaneous separation and cell disruption is shown in Figure 1.
The EFAC reactor was built in polyvinyl chloride, with a working volume of approximately 45 L, 60 cm high and diameter of 40 cm.
In its lower part, there is a polyvinyl chloride drainage port (1.3 cm of diameter) used for cleaning and/or sampling.The central cylinder, also made in polyvinyl chloride, had 20 cm diameter and the same height of the outside cylinder.At its bottom, a second polyvinyl chloride drainage port was installed with 1.3 cm diameter.The cathode and anode used were made of five 316-stainless steel bars, resistant to oxidative action, measuring 15 × 5 cm, thickness 0.2 mm, and spaced at 5 mm for each electrode.A direct current/alternating current converter (HY 125 Hobby, Hayama, Paraná, Brazil) was used to apply a voltage of 12 V and a maximum current of 5 A, generating a frequency band from 0 to 1.5 KHz, corresponding to a range of 0-4,000 cm -1 wave numbers.

Dewatering and cell disruption
Following phytoplankton diversity studies, one of the six systems evaluated was selected for dewatering and cell disruption experiments.
Samples were collected directly near the outlet of system B (1 facultative pond -Table 1).
The algal contents of the suspensions were measured before and after harvesting by EFAC in terms of turbidity and chlorophyll-a, after 40, 70, 120 and 140 minutes of batch operation.Floated microalgal cells were collected and lyophilized (L202,Liotop,São Paulo,Brazil) for subsequent lipid extraction.
The efficiency of EFAC for cell disruption was determined by the method described by Bligh and Dyer for lipid extraction (BLIGH & DYER, 1959).Dried biomass (500 mg) was diluted with 2.5 mL of methanol, 1.25 mL of chloroform and 1 mL of deionized water and then homogenized in a shaker for 20 min, after which 1.25 mL of chloroform and 1.25 mL of 1.5% sodium sulfate was added and the solution was shaken for 2 min for further homogenization.
The homogenized sample was centrifuged at 1,000 rpm for 2 min (Excelsa II 206 BL,Fanem,São Paulo,Brazil).The chloroform-methanol phase, which contains the extracted lipids, was separated by filtration using a funnel with mild suction followed by the evaporation of the solvent in an oven at 100ºC until constant weight was achieved.The mass of the lipid obtained from each sample was determined gravimetrically.

Data analysis
The efficiency of the EFAC method on cell disruption was determined based on the lipid yield, which was calculated by the ratio of total lipid extracted and the initial weight of microalgae.Statistical analyses of biomass separation results were performed using Microcal Origin 8.6 (OriginLab, Massachusetts, United States).
Simple bivariate correlation analysis was used to evaluate correlation between physicochemical parameters and the number of organisms that occur by genus (bloom) of microalgae detected.A Spearman correlation coefficient of 0.05 was used.

Phytoplankton diversity
A total of 21 phytoplankton genera, distributed in five classes, were identified on samples from the six stabilization ponds evaluated.
Five of the genera detected belong to the Cyanophyceae class, six to the Chlorophyceae, four to the Euglenophyceae, five to the Bacillariophyceae and one to the Zygnemaphyceae class (Table 2).
Although the samples were obtained from systems with different configurations (Table 1), phytoplankton diversity did not vary considerably.Samples from facultative ponds presented the same phytoplankton diversity, at the genus level, of other ponds such as maturation or facultative aerated ponds (Tables 1 and 2).
The class Cyanophyceae was the most diverse, presenting, in general, greater number of genera in comparison with other classes, except for system A, in which no genus of that class was identified, neither rare genera of the other classes nor a Euglena bloom were detected (Table 2).
Classes of Bacillariophyceae, Cyanophyceae, Chlorophyceae and Euglenophyceae were well represented in all studied effluents, with blooms of the genera Aphanocapsa, Planktothrix, Chlorella and Euglena (Table 2).These results are corroborated by the data of Patil et al. (1975), suggesting that these organisms are predominant only after the pond stabilization.These results are also in accordance with Palmer (1969), Padisák et al. (2003) and Bellinger & Sigee (2010), however they analyzed environments with high organic loads.In other reports, Di Bernardo (1995) concluded that cyanobacteria predominate in environmental conditions of low pH and low nutrients, which may explain, in this study, the blooms verified in systems A and D (Tables 2 and 3).Pham et al.
(2014) studied the spatial distribution of the algal community at different levels of BOD and also reported that the distribution was associated with the BOD concentration of the system.On the region of the pond with BOD of 74 mg L -1 , the community was dominated by motile algal Other genera found by that author in conditions of high organic loads were Scenedesmus, Nitzschia, Navicula, Oscillatoria and Stigeoclonium.Patil et al. (1975) noted in their studies that euglenoid organisms were better adapted to conditions of low organic matter and nutrients concentrations, in contrast to the results obtained by Palmer (1969).In the present study, the genera Euglena and Chlorella were found in relatively high frequencies in five of the six effluents evaluated.The absence of these genera in the waste stabilization ponds system F can be justified by the blooms of cyanobacteria in the genera Aphanocapsa and  Identification of microalgae from waste stabilization ponds and evaluation of electroflotation by alternate current for simultaneous biomass separation and cell disruption Eland et al. (2018) used molecular methods and flow cytometry to assess the cyanobacterial and eukaryotic communities longitudinally throughout a domestic wastewater treatment system and one mixed industrial/domestic wastewater system.Chlorella species and Planktothrix cyanobacteria dominated both treatment systems.Flow cytometry showed that the facultative and first maturation pond in the industrial system contained a lower proportion of photosynthetic organisms compared to the domestic system.This was reflected in the species richness data and low dissolved oxygen levels detected in the study.
The effluent from the system F presented the lowest ratio N:P (5.56±0.02)(Table 3), justifying the green algae absence on that environment (CUVIN-ARALAR et al., 2004).However, the system B presented bloom of Chlorella genus even for N:P lower than 20:1, contradicting those authors inferences.Organisms of the Bacillariophyceae class did not prevail in these conditions, which also disagrees with the latter investigation (CUVIN-ARALAR et al., 2004).
In order to determine which parameters could be related to the phytoplankton genera predominance in the study, correlations were performed between the number of organisms of each genus and the mean values of each parameter for all effluents.For the correlation analysis, only the six most representative of the 21 identified phytoplankton genera were considered (Tables 2 and 4).
The Spearman correlation coefficient values presented in Table 4 associate the parameters adopted as independent variable to the occurrence frequency of microalgae.It indicates the direction of those associations.The parameters nitrogen, BOD and temperature were the only ones to present a significant relation with the relative occurrence frequency of the genus Oscillatoria, in which N and BOD interfere negatively with the incidence of that genus (p<0.05) and temperature interferes positively (p<0.01).Therefore, increases in concentration of N and BOD induce the occurrence frequency of that genus, while an increase in temperate favors its appearance in the waste stabilization pond.For Chlorella, only the total phosphorus was related to the genus, negatively affecting its incidence (p<0.05),implying that increases in phosphorous concentration lead to a decrease number of cells of Chlorella.For all other organisms, although the Spearman coefficient has varied, there was no significant correlation and therefore any effect of the parameters on the listed genera could not be inferred (Table 4).

Simultaneous microalgae separation and cell disruption
The use of electroflotation (EFAC) on the effluent that presented bloom of Chlorella revealed high capacity for biomass harvesting.For instance, after 70 minutes of operation, turbidity removal reached values higher than 70% (Figure 2B).The application of EFAC for longer periods, 120 and 140 minutes, did not show any significant improvement on turbidity removal efficiency (p=0.08 and p=0.07, respectively).
The same behavior was verified for chlorophyll-a removal, in which after 70 minutes of operation the removal efficiencies were higher than 90% (Figure 2A), with no statistically significant difference upon comparison with 120 and 140 minutes (p=0.3).Therefore, 70-min operation was selected as the standard time for simultaneous microalgae separation and cell disruption assays.Azarian et al. (2007) evaluated electrolytic processes for microalgae removal from industrial effluents varying the power from 50 to 550 W. dm -3 .
At powers higher than 200 W.dm -3 , turbidity removal exceeded 80% for 10 minutes of residence time.However, there is risk of metal leaching when using consumable electrodes of aluminum at high power.Gao et al. (2010) evaluated microalgae harvesting by electrocoagulation-flotation, comparing the process efficiency using iron and aluminum electrodes, with a current density of 1 mA cm -2 , pH=4-7, and algae density=0.55× 10 9 -1.55 × 10 9 cells L -1 .These authors concluded that aluminum electrodes outperformed iron electrodes for algal removal and that electrolysis time required was shorter for aluminum electrodes.Identification of microalgae from waste stabilization ponds and evaluation of electroflotation by alternate current for simultaneous biomass separation and cell disruption compared a photoreactor (electro-oxidation and heterogenous photocatalysis) with pre-oxidants in a water treatment system.
The photoreactor reached higher turbidity removal efficiency (99.5%), higher COD removal (65.5%) and higher chlorophyll a (98.97%).It also removed higher concentrations of trihalomethanes (77.2 mg L -1 ) in comparison to other pre-oxidants.Fen et al. (2007) evaluated several interferents in electrodes for humic acid removal the continuous usage of aluminum.Operating with current density of within 60 minutes, 97.8% of 4.76 mA cm -2 and 1-cm space between the electrodes in acidic pH, this process reached 97.8% humic acid removal within 60 minutes.Benhadji et al. (2011) evaluated an electro-coagulation process with aluminum electrodes treating tannery.By applying a current density of 7.5 mA cm -2 for 45 minutes, they achieved up to 90% of BOD, COD, turbidity, chrome, iron and nitrate removal.
In the scenario of the present study, with non-consumable electrodes constructed of stainless steel, high efficiencies could be reached with a power of only 20.7 W.dm -3 .Therefore, it was possible to achieve high microalgal removal efficiencies using lower power than reported in literature, with a method that can avoid metals release to the medium, which represents cost savings in the separation process and ensures the viability of biomass for energy purposes.
The lipid content of a mixed microalgae community from a waste stabilization pond rich in Chlorella was 21.4±2.02%,which is close to reported values for pure cultures of representative species (LEE et al., 2010;PRABAKARAN & RAVINDRAN, 2011;WAHLEN et al., 2011;DE GODOS et al., 2011).Wahlen et al. (2011) evaluated the lipid content of microalgae collected from a wastewater treatment plant using ultrasound sonication and lipid extraction using organic solvents and obtained a 14.4% lipid yield.
Several studies have been conducted to compare methods traditionally used as pretreatment for cell disruption of microalgae derived from either pure or mixed cultures.Koberg et al. (2011) compared the efficiency of microwaving (5 min at 70% power (cycle mode of 21 s on and 9 s off) and ultrasound (20 kHz, 40% amplitude for 5 min) for disruption of microalgae of the genus Nannochloropsis with simultaneous extraction of lipids by the organic solvents.These authors achieved percentages (in biodiesel) of 10.7% in the control group, 18.9% in the group pretreated under ultrasound radiation and 32.8% in the group pretreated under microwave radiation.The authors related the results obtained to the higher temperature increase in microwave radiation process than the provided by sonication process, carrying the greatest cell disruption and the increased releasing of lipids for conversion to biodiesel by transesterification method.Lee et al. (2010) compared five cell disruption methods: autoclave (125ºC with 1.5 MPa for 5 min), bead-beating (2,800 rpm for 5 min), microwave (100ºC and 2,450 MHz for 5 min), ultrasound (10 kHz for 5 min) and osmotic shock (10% NaCl solution with a vortex for 1 min and maintained for 48 h).The methods were applied to pure cultures of Botryococcus sp., Chlorella vulgaris and Scenedesmus sp.The efficiency of each method was measured by the achieved yield in the lipid extraction using chlorophorm and methanol.The authors obtained lipid percentages ranging from 5.4 to 11.9% using autoclaving, 10 to 28.6% using microwaving, and 6.1 to 8.8% using sonication.Prabakaran & Ravindran (2011)   Regarding the application of EFAC reactor, this paper reported the first attempt of an efficient method for simultaneous microalgae separation and cell disruption.Thus, there is no data in literature that allow a comparative study, hampering an elaborated discussion.

CONCLUSIONS
The current paper described the first attempt at using an EFAC reactor for simultaneous microalgae separation and cell disruption, showing promising results for the application of EFAC to produce biodiesel from microalgae grown on waste stabilization ponds.
Phytoplankton genera predominant in the studied systems belonged to Chlorophyceae, Cyanophyceae and Euglenophyceae classes.These classes of microalgae normally present relatively high lipid content and predominantly saturated fatty acids, which can be advantageous in the production of biodiesel.
The lipid contents obtained with microalgae from stabilization ponds treating domestic wastewater resemble those yields obtained from microalgal biomass growth in conventional processes and reported in literature.
Regarding the application of EFAC reactor, as far as we are concerned, this is the first attempt of an experiment on simultaneous microalgae separation and cell disruption, with promising results for application in systems that produce this biomass for biodiesel production.
species such as Euglena and Cryptophytes.When the BOD concentration decreased, the algal community was more evenly distributed and nonmotile species such as Chlorophytas and Chrysophytes were also present.Genera Euglena, Chlorella and Chlamydomonas are considered the most tolerant to pollution by high organic matter compounds and present great adaptation capacity to climate change (PALMER, 1969).

Figure 1 -
Figure 1 -Schematic diagram of the electroflotation by alternate current (EFAC) reactor used for simultaneous biomass separation and cell disruption.
| v.24 n.1 | jan/fev 2019 | 177-186Florentino, A.P. et al.    obtained from waste stabilization ponds, as the ones used on the present study, offer higher probability of application, while they remove the purification step from the process.Wang et al. (2015) evaluated the cell disruption degree of a culture of Neochloris oleoabundans pretreated under ultrassonication (power of 400, 600 or 800 W for 50 min), high-pressure homogenization (operating pressure of 40, 50, 60, 70 or 80 MPa), enzymatic hydrolysis (cellulase and / or papain) and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication (papain+cellulase, 600 W for 30 minutes, 80 Mpa).The highest disruption degree, up to 95.41%, was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization.The authors obtained 36% of lipid yield with the best method for cell disruption in comparison to the control group -with no pretreatment, which counted with 10% lipid yield.Recently,Phong et al. (2018)  investigated the effect of ultrasound (37 kHz for 1,200 s) on the cell disruption of Chlorella sorokiniana and Chlorella vulgaris for protein extraction.The authors observed that the sole application of ultrasonication for both strains provided two to three-fold increase in the protein yield compared to their corresponding control groups.Similar reults were obtained byParimi et al. (2015), who found that ultrasonication (20% maximum power for 60 min) resulted in higher protein recovery (69.9%) in the supernatant than that of the control group, without pretreatment step.The same authors performed autoclaving as pretreatment (at 121°C with 103.4 kPa for 30 min), and high-pressure homogenization involving two passes through a high-pressure homogenizer (at 103.4 Mpa), obtaining 29 and 83.5%, respectively.A study conducted byAraujo et al. (2013) observed that the application of ultrasonication (40 kHz with ultrasonic intensity of 29.7 W L -1 or 2.68 W m -2 ) in the extraction process was able to improve the extraction efficiency (52.5%) of intracellular compounds in microalgae.

Table 1 -
Configurations of the waste stabilization ponds systems selected.

Table 2 -
Semi-quantitative distribution of microalgae genera found in the studied stabilization ponds systems.

Table 3 -
Values of the physic-chemical parameters analyzed in the effluents characterization.