WO2017106941A1 - Method for treating waste water using a culture of polysaccharide-excreting micro-algae, and use of micro-algae - Google Patents

Abstract

The present invention relates to a method for treating waste water comprising a suspension of fine particles of solid fuels, using a culture of micro-algae which produce extracellular polysaccharides. The invention further relates to the use of polysaccharide-excreting micro-algae for treating waste water comprising a suspension of fine particles of organic and inorganic materials from solid fuels.

Description

 RESIDUAL WATER TREATMENT METHOD THROUGH CROP OF MICROALGAE EXCRETING MICROALGAS AND MICROALGAL USE

FIELD OF INVENTION

 The present invention relates to the wastewater treatment method comprising fine particles of suspended solid fuels by cultivating extracellular polysaccharide producing microalgae.

TECHNICAL STATE

The need for cement by today's society is high due to the increase of urban areas, infrastructures and the consequent growth of civil construction. Unfortunately, its production causes a risk of environmental contamination. This is due to the high amount of gases such as C0 ₂ and N0 ₂ that the cement industry releases into the environment. These gases come from the burning of solid, liquid or gaseous fuel constituents in the kilns for cement production. Thus, the cement industry demands large quantities of fuel, among which oil coke stands out for its low cost.

 Petrocoque is a solid fuel widely used in the cement industry because it has a high fixed carbon content, low ash content, high calorific value and high chemical stability. It is neither reactive nor explosive (PETROBRAS, 2014). This product reaches the industries by road, in trailers, which will be reused for the transportation of other products, and therefore its interiors must be washed with water, which together with the rainwater forms the residue called black water, where the Petroleum coke remains insoluble in fine grains. Fine fractions of other solid fuels such as coal, charcoal and bituminous coal (anthracite) can also generate petrocoque-like wastewater.

 This waste cannot be disposed of in the recipient body by industries, as it contains traces of oil and other contaminants, and may be harmful to the environment. Petroleum coke, also called petrocoque, is a solid material resulting from the distillation of petroleum obtained from the cracking of heavy residual fractions (PETROENERGIA, 2014).

 Regarding the toxicity of petrococcus to the environment, it was thought to be inert, and was even thought to be considered as a remediation agent for petroleum contaminated soils (FEDORAK et al; 2006; NARAYANAN et al; 1997). However, recent studies have shown that petrocoque is not inert and causes effects on terrestrial plants, aquatic animals and microorganisms (MCKEE et al., 2014).

Literature data show that the presence of petroleum coke in the environment can lead to increased concentration of trace elements in soil and water. (Chen et al., 2010). The presence of higher concentrations of vanadium and nickel are indicators of the presence of petroleum coke in the medium (ZHAO et al., 2002); These and other trace metals normally exist in aqueous media in the form of dissolved ions and have great potential to cause adverse effects on microorganisms if present in amounts above those usually found and in free form, not complex to organic materials (MORGAN, 1987; PEIJNENBURG et al., 1997; LOMBARDI et al., 2002; TONIETTO et al., 2014).

 The literature shows several methodologies for the treatment of industrial waste and effluents. LOPES et al, (2014) used synthetic mineral analogs for the removal of metals, organic compounds and petroleum residues from soils and aquatic media. However, this and other conventional technologies, with physicochemical treatments, have high cost and can generate toxic waste to the environment (CHANDRA et al, 2013).

 Bioremediation, a technology used for the recovery of degraded environments, is based on the natural capacity of microorganisms to degrade waste (MILIC et al. 2009), and offers great potential for decontamination of natural environments and industrial effluents. Several studies have confirmed the ability of microalgae to produce polysaccharide excretes capable of binding to metals by remediating effluent (TONIETTO et al., 2014; LOMBARDI and VIEIRA, 1999; LOMBARDI and VIEIRA, 2000). There is also the possibility of bacteria and fungi acting as hydrocarbon degraders (CHANDRA et al.,

2013).

 Studies in the area of microalgae biotechnology have already proven the ability of these photosynthetic microorganisms to remediate arsenic, lead, chromium, cadmium, uranium and dyes contaminated waters (KHANI et al, 2008; JASROTIA et al, 2014; PARAMESWARI et al, 2010 ), treat other industrial waste (HENDE et al,

2014), in addition to its role in wastewater remediation of sewage treatment plants (MARCHELLO et al., 2015). In wastewater recovery, microalgae are co-cultivated with other microorganisms such as bacteria. These bacteria are mostly heterotrophic and aerobic, which generates a synergistic mechanism where algae produce 0 ₂ that is used by bacteria to mineralize organic matter (MO), which in turn release the end degradation product C0 ₂ , used by microalgae. This process releases the mineral nutrients associated with OM that will also be used by microalgae. Thus, a microbial consortium is created that acts synergistically involving the algal and bacterial community. This strategy can reduce the high cost of mechanical aeration in algal crops. As typically photosynthetic beings (oxygenic photosynthesis), microalgae have the power to mitigate the C0 ₂ reducing problems related to the Greenhouse Effect, which is responsible for the increase in the earth's temperature, with disastrous consequences for society. It is known that the greenhouse effect can lead to problems such as melting polar ice caps, rising ocean levels, and extreme climate change (ESSAM et al, 2006; ESSAM et al, 2013; MUNOZ & GUIEYSSE, 2006; SOON et al., 1999).

The largest anthropogenic C0 ₂ emissions result from the burning of fossil fuels and cement production (EPA, 2010). The production of cement accounts for 5% of the global emissions unnatural C0 ₂ (Borkenstein et al, 201 1,. HASANBEIGI et al, 2012;. WBCSD 2009). The International Energy Agency predicts that emissions of C0 _2, from energy production will reach 38 billion tons per year in 2030, a rate 70% higher than 2002 levels (IEA, 2002). Due to this alarming rate, in 1997, more than 170 countries signed the Kyoto Protocol, which required signatory countries to reduce emissions of harmful gases into the atmosphere, known as greenhouse gases (GHGs) (UNFCC, 2012).

 In addition to bioremediation capacity, microalgae can generate value and save money based on this, as their cultivation results in biomass rich in biomolecules of interest, such as lipids, proteins and carbohydrates. Among the various applications of algal biomass, its use is cited in the food, feed, dye, pharmaceutical and cosmetic industries, as well as in the biochemical industry, for the production of bioplastics and biofilms, as well as chemicals such as biofertilizers and antioxidants (SAFI et al, 2014).

 Due to the depletion of fossil fuel reserves, the rising price of its derivatives and concern about global warming, attention has been paid to the bioenergetic capacity of microalgae. The production of biofuels through lipid accumulated by algae in the form of triglycerides (CHISTI, 2007) and aliphatic hydrocarbons (ROCHA et al, 2014). According to CLARENS et al. (2010) Energy produced from microalgae has the potential to have a lower impact on the environment compared to energy from plants (CLARENS et al, 2010).

Some microalgae like Chlorella sp. They are robust and capable of adapting to a variety of freshwater and saltwater environments, from frozen to desert, pristine or contaminated, such as municipal, industrial and agricultural sewage and industrial co-products such as vinasse and soybean whey. (MARCHELLO et al, 2015; MITRA et al, 2012). Algae cultivation in sewage treatment plants and industries can offer several benefits such as the removal of nutrients from the wastewater, the biomass energy production and generated fixing C0 ₂ (Akerstrom et al., 2014).

 Some forms of wastewater treatment using microalgae are described in patent documents such as US 2015175457, US 2009294354, US 2003213745, US 5476787, EP 2093197 and US 2013061517. Additionally, US 2009294354, US 2003213745, US 5476787, EP 2093197 and US 2013061517 deal with the use of wastewater for biomass production.

In addition to proposing the treatment of effluents containing petroleum residues, document CN 1031 12993 also mentions the fixation of CO ₂ by microalgae, and the algae are later used as oil source in biodiesel production. This document also reports that microalgae has high stress resistance, high oil content and high degradation function of petroleum hydrocarbons.

 US 3763039 and US 4966713 involve wastewater treatment employing microalgae that act as flocculating agents of waste present in water.

 The document CN 103881923 refers to the cultivation of microalgae using the effluent generated in the coking of coal, together with seawater.

The invention described in US 2013174734 has as its primary objective the reduction of particulate matter present in water or air or other means, such particulate material being obtained from mining activities. An important feature in US 2013174734 is that the agglomeration of particulate material is due to negatively charged exopolysaccharides (EPS) which are obtained from isolated, purified and used bacterial or algal cultures. It is further specified that if the particulate material is present in the air, the EPS in solution will be sprayed, or alternatively, the EPS may be immobilized on the filter and the air forced through the filter. In this situation the particulate material will be aggregated. If particulate material is present in water, it will be mixed with another negatively charged EPS-containing solution and the particulate material will agglomerate. US 2013174734 differs from the present invention in obtaining, origin and characteristics of the excreted polysaccharide. As for obtaining: it is proposed to cultivate microalgal directly in the wastewater (black water), which will be the receptor body of the polysaccharide excreted by the algae that grow there, and the suspended solid particles have very different characteristics of the particulate from the algae. mining activity. Regarding the origin: in this, it is proposed the use of polysaccharides excreted exclusively from microalgal origin, microalgae capable of performing oxygen photosynthesis. Regarding the characteristics of exopolysaccharide: In this, it is proposed the use of microalgal excreted polysaccharide whose composition is complex containing about 70% of neutral monosaccharides and only about 12 - 15% of acid monosaccharides, thus conferring neutrality in general. In the present invention, the excreted polysaccharide acts as a binder of particles, which stick together and with the polysaccharide, settle to the bottom, being removed from the liquid phase, where the growing microalgae remain.

 Compared to US 2013174734, whose EPS is used at a concentration of 1%, the present invention has the advantage of dispensing with any processing or purification step of the excreted polysaccharide as it is directly excreted by microalgae in the culture medium. eg wastewater, where it will exert its action.

 In all the documents presented here the wastewater treated by microalgae or used as a culture medium for its growth is domestic or industrial effluents or water from the petroleum or coke production process. Such effluents contain residues of mineral elements and organic matter, the former being absorbed by cells, while the latter may (or may not) be degraded by microalgae, causing water treatment and algal biomass production. With the exception of US 2013174734, effluents treated or used as a microalgae culture medium have dissolved material in water and no suspended solids as described in the present invention. On the other hand, while US 2013174734 deals with airborne or waterborne particles, these particles are derived from mining activity and do not consist of solid fuel residues such as petroleum coke, mineral and vegetable coal and anthracite.

 None of the prior art documents describe the treatment of wastewater containing fine particulates of suspended solid fuels by cultivating microalgae responsible for the excretion of polysaccharide carbohydrates in the culture medium, which will agglutinate said particulates. The agglutination of the particulate material allows its exclusion (sedimentation) from black water, resulting in clean water and algal biomass. Since the residues remain agglutinated by the excreted algal polysaccharides and are not absorbed by the microalgae, the obtained biomass can still be used in various applications, such as oven burning, biofuel, animal feed, among others already mentioned.

 Thus, the present invention aims to:

- the treatment of wastewater with the presence of suspended fine particulate organic and inorganic material from solid fuels, known as black water; - the reuse of treated black water after microalgal cultivation;

 - the reuse of black water as a microalgal cultivation medium

- reuse of algal biomass and solid fuel residues that, collected, can be reused in an industry furnace;

 - reuse of algal biomass for various applications, as it will be collected separately from agglutinated and sedimented particles;

- fixing of C0 ₂ by microalgae used for black water treatment; resulting in the recovery and valuation of wastewater (black water) from industries that use solid fuels, especially that generated in the cement industry, thermoelectric plants, steel mills, etc.

 BRIEF DESCRIPTION OF THE INVENTION

 The present invention is a method of treating wastewater by cultivating polysaccharide excretory microalgae comprising the following steps:

 (a) collecting in a wastewater treatment tank comprising, in suspension, fine particulates of organic and inorganic material from solid fuels;

 (b) inclusion of nutrients in wastewater;

 (c) inclusion of the polysaccharide excretory microalgae inoculum in the wastewater obtained in step (b);

 (d) separation of the fine particulates by agglutination with the excreted polysaccharides and subsequent decantation of the agglutinated particulates at the bottom of the tank;

 (e) microalgae collection; and

 (f) collection of treated water.

 The invention further relates to the use of polysaccharide excretory microalgae in wastewater treatment comprising, in suspension, fine particulates of organic and inorganic material from solid fuels.

BRIEF DESCRIPTION OF THE FIGURES

 Figure 1: Samples of black water with nutrient addition and inoculated with Chlorella vulgaris. A: without particulates of agglutinated solid fuels; B: with particulates of agglutinated solid fuels.

Figure 2: Black water viewed under an optical microscope. Different species of microalgae present in BG-1 nutrient-enriched black water 1. A: circular green algae in diameter about 2,5 μηι; B: cluster of green algae and C: shows a species of microalgae of larger diameter, approximately 20 μηι. Figure 3: Black water viewed under an optical microscope. Note the presence of

Bacillariophyceae in both samples, besides inoculated C. vulgaris.

 Figure 4: Microalgal community growth quantified by fluorescence variation as a function of cultivation time in treatments inoculated with Chlorella vulgaris (SN = no nutrient increase; CN = nutrient increase).

 Figure 5: Variation of pH as a function of cultivation time (SN = no nutrient increase; CN = nutrient increase).

 Figure 6: Growth rates obtained for all treatments performed. Light: A, B, C, D; Dark: E, F, G, H. With nutrients (CN): A, D, F, H; Nutrient-Free (SN): B, C, E, G. Inoculated Chlorella vulgaris: C, D, G, H.

 Figure 7: Concentration of intracellular carbohydrates in exponential growth phase and at the end of the experiment in inoculated cultures. A = Light SN exponential phase; B = SN light final phase; C = Light CN exponential phase; D = Light CN final phase. Inoculated experiments.

 Figure 8: Protein concentration at exponential growth phase and at the end of the experiment in inoculated cultures. A = Light SN exponential phase; B = SN light final phase; C = Light CN exponential phase; D = Light CN final phase.

DETAILED DESCRIPTION OF THE INVENTION

 The present invention is a method of treating wastewater by cultivating polysaccharide excretory microalgae comprising the following steps:

 (a) collecting in a wastewater treatment tank comprising, in suspension, fine particulates of organic and inorganic material from solid fuels;

 (b) inclusion of nutrients in wastewater;

 (c) inclusion of the polysaccharide excretory microalgae inoculum in the wastewater obtained in step (b);

 (d) separation of the fine particulates by agglutination with the excreted polysaccharides and subsequent decantation of the agglutinated particulates at the bottom of the tank;

 (e) microalgae collection; and

 (f) collection of treated water.

Waste water of the present invention refers to water with the presence, in suspension, of fine particulates of organic and inorganic material from solid fuels that will be burned in industrial kilns, especially those used in the cement, thermoelectric, steel, etc. These suspended materials include petroleum coke, charcoal and charcoal. mineral, coal or coke grade steel, anthracite and / or mixtures thereof in any proportion. This water may originate from rainwater, which sweeps away deposits of material and / or flush water from places that served as deposits for such fuels. It is called black water because of its dark color due to the fine particles of fuels mentioned above.

 Such fine particles of solid suspended fuels are those particles that remain suspended in water even though it has gone through the settling process, which separates the larger particles from solid fuels. In this way, smaller and lighter particles that do not go to the bottom of the settling tank remain and are removed according to the method described herein by agglutination with algal polysaccharides and settling at the bottom of the tank.

 Accordingly, the fine particulates of organic and inorganic material originating from solid fuels suspended in wastewater according to this invention may be coal, charcoal, petroleum coke, coal or steel grade coke, anthracite or mixtures thereof. Other rain-borne materials such as grains of clay and / or sand may be present in the wastewater to be treated.

 Some algal groups are important polysaccharide excrectors, such as the group of Cyanophyta (blue algae), Heterokontophyta (Bacillariophyceae, diatoms) and Chlorophyta (green algae). Chlorophyta division, among others, includes robust microalgae for a wide range of environmental conditions and chemical agents such as the genera Chlorella, Scenedesmus, Desmodesmus, Monoraphidium (VIEIRA et al, 2008).

 In accordance with the present invention, polysaccharide excreta microalgae are chosen from the groups of Bacillariophyta, Chlorophyta and Cyanophyta or any other polysaccharide excretory microalgae, depending on the purpose of the biomass. Particularly, microalgae are chosen from those of the genera Chlorella, Scenedesmus, Desmodesmus and Monoraphidium; preferably Chlorella vulgaris. In the present invention the microalgae Chlorella vulgaris is used because it is robust and withstands variations of environmental conditions, which allowed its growth in wastewater. Its later use as animal feed is possible as the microalgae will be separated from the fine particulate material.

For rapid growth of microalgae prior to step (c) of inclusion of the microalgae inoculum, wastewater used as culture medium is enriched with major and minority inorganic nutrients necessary for the development of polysaccharide excretory microalgae. Nutrient Examples Major compounds which may be used in accordance with the present invention are: nitrates, phosphates, magnesium, calcium. Minority nutrients can be chosen from iron, manganese, copper, molybdenum, cobalt and zinc.

 Thus, after the addition of nutrients and inoculation of the polysaccharide excretory microalgae into the wastewater to be treated (steps (b) and (c) of the process described herein respectively), algal culture and growth are initiated, resulting in excretion. polysaccharides responsible for the agglutination of the fine particles and consequent decantation of such agglutinated particles, which may or may not be adhered to the bottom of the treatment tank.

In accordance with the method described herein and in order to enable microalgae growth, steps (c) and (d) occur under light intensity ranging from 100 to 300 μηιοΙ m ^"2 s ^"1; preferably 150 μηιοΙ m ^"2 s ^{" 1} . However, in the natural environment, the light intensity varies according to local climatic conditions, and may reach up to 1,500 μηιοΙ m ^"2 s ^{" 1} of incident sunlight, without affecting algal growth. These steps (c) and (d) occur in a photoperiod similar to the natural one, 12 hours light and 12 hours dark.

 The wastewater treatment method according to the invention comprises agglutination of the fine particulate of organic and inorganic material from solid fuels that are suspended in the wastewater and their decantation at the bottom of the treatment tank.

 The fine particulate agglutination of organic and inorganic material from solid fuels suspended in wastewater is due to the excretion of polysaccharides by microalgae, which act as cementing material. These fine particulate agglutinates, together with the material excreted by the microalgae, naturally decant at the bottom of the treatment tank, leaving above the water treated with the microalgae.

 Polysaccharide excretion is a normal process during algal growth, but may be increased or decreased depending on the nutritional status of the microalgae (VIEIRA et al, 2008).

The materials excreted by microalgae may vary in composition and, for them, have several functions. According to the literature, excreted polysaccharides may be involved in cell mobility, stabilization, formation of cell clusters and adhesion of cells to surfaces, depending on the algal species. Overall, the investment by microalgae in the excretion of polymeric substances is justified by the protection that these substances offer to cells (MYKLESTAD, 1995; LOMBARDI et al, 2005). For example, they protect the cell against desiccation, attraction of nutrients close to the cell surface, against excess solar radiation, among other functions.

 It is known that the composition of microalgae excreted polysaccharides may vary among the algal groups, however their carbohydrate base is common to all. Chlorophyta algae are less protein and more polysaccharide compared to cyanobacteria (TONIETTO et al., 2014; LOMBARDI and VIEIRA, 1999). On the other hand, the Bacillariophyceae group may have a certain hydrophobic character (VIEIRA et al., 2008). Microalgal polysaccharide is usually made up of neutral carbohydrate monomers such as fucose, arabinose, galactose, glucose, mannose, rhamnose and xylose and / or acids such as galacturonic and glucuronic acids (LOMBARDI and VIEIRA, 1999). Carbohydrates make up about 70 - 80% of the excreted polysaccharide (LOMBARDI and VIEIRA, 1999; VIEIRA et al, 2008); proteins can reach 15-20% (LOMBARDI and VIEIRA, 1999), while lipids can reach 1 - 2% (PARRISH and WANGERSKY, 1987; LOMBARDI and WANGERSKY, 1991).

 Since the fine particulate is agglutinated with the polysaccharides excreted by the microalgae, we have the water treated with the microalgae.

 Following is the separation and collection of microalgae, obtaining, on one side, the treated water and free of fine particulate organic and inorganic material from solid fuels, and on the other, the algal biomass.

 Treated water free of fine particulate solid fuels and microalgae can be used in a variety of applications, such as reusing it to wash solid waste haulers, cleaning the surrounding area, washing the solid waste stock yard, irrigation gardens, feeding photobioreactors, among others.

 On the other hand, the present invention also enables the use of cultivated and collected microalgae according to the method of the invention. This algal biomass can be used in various applications, such as biofuel production, burning in factory furnaces, use as animal feed, among others. However, to define the use of algal biomass, one should choose the most suitable species for the purpose. For example, Chlorella vulgaris has high protein content (CHIA et al., 2013a; 2013b; 2015) and is therefore indicated for such application. Scenedesmus sp produces aliphatic hydrocarbons (ROCHA et al., 2014) and may be an interesting species for biofuel production.

Optionally, the microalgae collection step (e) may comprise a prior flocculation step followed by the microalgae decantation. Thus, the fine particulate agglutinated with the polysaccharides excreted by microalgae together with the algal biomass, thus obtaining the treated water in the supernatant.

 Microalgae flocculation can be achieved by adjusting the residual water pH to an ideal algal flocculation pH; or by the use of flocculating agents such as aluminum sulfate and aluminum chlorohydroxide; or by any other known method of the art for microalgae flocculation. Preferably, the pH adjustment of the wastewater to the range between pH 10 and pH 11 is used, and after flocculation and decantation of the microalgae, the treated wastewater is adjusted to its pH again according to the standards established by the control bodies. control and supervision of the emission of liquid effluents into the environment.

 The treated water obtained from the method comprising the prior stage of microalgae flocculation can also be used in various applications, such as those previously reported. Decanted material, including fine agglutinated particulate matter and microalgae, can be reused as fuel.

 The present invention deals with the use of polysaccharide microalgae excreta as effective agents in wastewater treatment comprising, in suspension, fine particulates of organic and inorganic material from solid fuels, which treatment is achieved, since wastewater is used as culture medium for polysaccharide producing microalgae. This polymer acts as a fine particle binder that, with larger size, decant at the bottom of the treatment tank. Due to the type of polysaccharide excreted, such fine agglutinated and decanted particles may or may not remain adhered to the bottom of the treatment tank.

EXAMPLES

 The analyzes presented below show the use of wastewater from the cement industry for microalgae cultivation. To this end, the following analyzes were continued:

 - cultivation of microalgae community, including inoculated Chlorella vulgaris, under various experimental conditions, all in black water; - Quantification of algal growth in black water plus nutrients;

- Quantification of algal growth in black water without the addition of nutrients;

 - observation of agglutination of particulate matter.

- Quantification of the biochemical composition of biomass generated in black water through the determination of carbohydrate and protein concentration in cells. MATERIAL AND METHODS

Origin of black water

 The petroleum coke, charcoal, anthracite and mineral coal fuels used in combustion in cement industries in Brazil reach the industries by road and for the trucks that transported them to be reused they must be washed. Such washing results in an aqueous residue which is called black water. This liquid residue containing fuel particles is stored in settling tanks as it cannot be disposed of. The black water samples used in this study were collected from the first level of the settling tank, where there was a higher amount of suspended particulate matter.

 Black Water Analysis - BTEX

 The black water used was analyzed for the determination of organic compounds (benzene, toluene, ethylbenzene and xylene), commonly known as BTEX. The determinations were performed by the company Global Análises e Consultoria, located in São Carlos (SP, Brazil) shortly after the wastewater arrived at the laboratory. BTEX analyzes were performed by solid phase microextraction followed by gas chromatography coupled to mass spectrometry (SPME-GC / MS). The sample preparation phase (SPME) was followed by the methodology described in VALENTE and AUGUSTO (2000) with modifications by QUEIROZ (201 1).

 Black water testing as a culture medium for microalqas

 Experimental planning

 The wastewater (black water) was transported from the cement industry to the laboratory in polypropylene gallons. In the laboratory, black water was packed in smaller bottles, some of these bottles were frozen, others were refrigerated for immediate use, ensuring the maintenance of the microorganisms present.

 Aiming the treatment of black water through microalgal cultivation, several tests were performed:

 - nutrient-enriched black water from BG-11 medium (ANDERSEN, 2005) inoculated with Chlorella vulgaris microalgae and kept under light (Light - CN) and in the dark (Dark - CN);

 - pure black water, without added nutrients, inoculated with Chlorella vulgaris microalgae and kept in light (Luz - SN) and dark (Dark - CN);

- pure black water, without nutrient increase kept in light (Light - SN) and dark (Dark - SN);

Assays were performed in 690 mL cell culture flasks containing 250 mL of black water in experimental triplicates. In tests where the black water was enriched with nutrients from culture medium BG-1 1, the following amounts were added: NaN0 ₃ (1.5 g L ^"1 ); K ₂ HPCy3H ₂ 0 (40 mg L ^{" 1} ); MgSCy7H ₂ 0

(75 mg L- ¹ ); CaCl ₂ -2H ₂ 0 (36 mg L- ¹ ); NaCO ₃ (20 mg L ^"1 ) FeCl ₃ -6H ₂ 0 (3.15 mg L ^{" 1} ); citric acid (6 mg L ^"1 ) and 1 mL of micronutrient solution composed of H ₃ B0 ₃ (2.86 mg L ^{" 1} ), MnCl ₂ -4H ₂ 0 (1.81 mg L ^_1 ), ZnS0 ₄ - 7H ₂ 0 (0.22 mg L ^"1 ), Na ₂ Mo0 ₄ -2H ₂ 0 (0.39 mg L ^{" 1} ),

CuSCy5H ₂ 0 (0.08 mg L ^"1), Co (N0 _{3) 2} 6H ₂ 0 (0.05 mg ^L-1).

The experimental replicates were kept under light intensity of 130 μηιοΐ ητν ¹ , with natural photoperiod (approximately 12: 12h light: dark) at 23 ^and C, and the temperature may vary between 23 - 29 ^and C.

Chlorophyll Fluorescence

 In vivo fluorescence (Turner - Trilogy, USA Model 7200-043 Fluorimeter) was used as a measure of chlorophyll a concentration, a parameter that relates to photosynthetic microalgae biomass. This methodology was used to verify the population density of the samples, because in the case of black water as a culture medium, it was not possible to count cells under an optical microscope to quantify living biomass, due to the intense presence of sediment in the medium.

 Hydrogen Potential - pH

 To measure the pH of the samples a Logen® Scientific pH meter was used. The pH is an important parameter because the cultivation of microalgae modifies the pH of the medium. Normally by cultivating photosynthetic microalgae the pH of the medium increases and may reach pH 11 if the medium is not buffered.

 Total protein quantification

 Total protein extraction and quantification was performed using the protocol described by BRADFORD (1976) and RAUSCH (1981). Quantification is done by spectrophotometer and calibration curves were obtained at wavelengths of

595 nm. Details of the procedure can be found in CHIA et al. (2015).

 Total Carbohydrate Quantification

Total carbohydrate quantification was performed based on the technique described by ALBALASMEH et al. (2013) through spectrophotometry. This technique is based on the reaction of sulfuric acid with algal biomass. For calibration curves glucose was used as standard and intracellular carbohydrates were quantified as glucose equivalent ^ g mL ^"1 ). Details of the method can be found in CHIA (2012).

 Growth rate

Algal growth rates were obtained from population density values through graphical analysis by plotting the natural logarithm of fluorescence values as a function of experimental time in days. During growth Exponentially, this graph results in a line that can be adjusted by linear regression. The angular coefficient of the adjustment represents the specific growth rate, whose unit is day ^"1 .

Sample preparation and visualization

 Samples of black water were prepared on slides for observation under the optical microscope (Nikon Digital Sight DS-U3) so that their visualization and photography to verify the particulate material became possible.

RESULTS AND DISCUSSION

Organic Compounds (BTEX)

 Table 1 shows the results obtained from the BTEX analysis at the concentrations present in the black water used as the basis for the microalgae culture medium.

 Table 1 Concentration of hydrocarbons present in black water used as a culture medium for microalgae.

Hydrocarbons Concentration ^ g L ¹ )

 Benzene ND

 Toluene ND

 m-Xylene 0.49

 p-Xylene 0.55

 o-Xylene 0.39

 Ethylbenzene 3.77

 ND = Not detected.

According to PENG et al. (2014), the toxicity of BTEX to microalgae cells and inhibition of chlorophyll synthesis follows the order xylenes>ethylbenzene>toluene> benzene. In the present analysis, the hydrocarbons considered more toxic to algae were those detected in black water. However, a study with the freshwater microalgae Selenastrum capricornutum showed that xylenes were toxic to this algae at a concentration of 3.9 mg L ^"1 or higher and for ethylbenzene 4.7 mg L ^{" 1} (HERMAN et al. , nineteen ninety). For Chlorella pyrenoidosa algae, nitrobenzene was toxic at concentrations of 28 mg L ^"1 and 2-nitrotoluene at 22 mg L ^{" 1} (RAMOS et al., 1999). In the results of this study, the compounds showed no toxicity to the inoculated microalgae Chlorella vulgaris.

Bioassays

Black water, when viewed under the microscope, had only dark sediments (Figures 1A and 1B), some protozoa, rotifers and microalgae, many of which were filamentous. In the dark trials, nutrient-enriched or black water remained unchanged and the sediments remained the same size and appearance (sandy) after 1 month of incubation. However, in black water with or without nutrients, but kept in light, changes in sediments were observed. The sediments agglutinated and viscously deposited and adhered to the bottom. vial (Figures 2A, 2B and 2C). Microalgae-excreted polysaccharides are known to have this property of sticking or agglutinating particulates (VIEIRA et al., 2008). Under the microscope, it was observed that not only the inoculated microalgae grew in the crops, but also others that were already in black water. Filamentous microalgae, coccoids, and Chlorella vulgaris were observed in black water.

 Due to the versatility and robustness of Chlorella vulgaris microalgae, it was chosen for inoculation in black water. When viewed under an optical microscope (Figure 1), it was found the presence of free C. vulgaris in the medium and also in the particles after 15 days of cultivation. The sediments present in the black water were agglutinated and increased in size from sandy to viscous granules. Diatom microalgae were also visualized (Figure 3), suggesting the presence of silica in black water, as these algae need this compound for their development.

 Black water assays confirmed the viability of photosynthetic microalgae growth in this residue by increasing chlorophyll a fluorescence and observations under a microscope. It is concluded that the increase of nutrients is essential to ensure higher biomass and higher population growth compared to non-nutrient black water. Figure 4 shows the highest growth for the nutrient black water test inoculated with C. vulgaris and kept in the light.

 Figure 5 shows the pH variation according to the experimental time. Algal growth was marked by pH 9-10 of the cultures kept under light.

 In the presence of light, but without nutrients, black water supported limited growth of microalgae, a fact confirmed by chlorophyll a fluorescence values (lower than in the presence of nutrients) and pH values, which decreased over time. In the absence of light, there was no microalgae growth and the pH was reduced to 7.5. Without inoculation of C. vulgaris, but added nutrients and incubated in light, the growth of microalgae in black water was slow, requiring 80 days to increase fluorescence, which confirms the importance of inoculation of C. vulgaris.

 There is a large difference in light and dark treatments. While in the light increasing fluorescence values were obtained since the beginning of cultivation, as observed in Figure 4, in the dark, lower fluorescence values of chlorophyll a were obtained. The pH values of the samples in the light were higher than the pH of the samples in the dark throughout the experiment (Figure 5).

Black water maintained in the dark, enriched or not with nutrients, obtained low fluorescence values, while light treatments showed fluorescence values characteristic of the growth of photosynthetic microorganisms. Analyzing the pH values of the treatments, it can be observed that in the experiments with nutrient-enriched black water and kept in the light, the pH remained more alkaline than the tests with non-enriched black water, especially the treatments kept in the light. This demonstrates increased algal growth in black water plus nutrients and kept in light. The pH is a factor of great importance in algae cultivation, because it determines the solubility of C0 ₂ and mineral nutrients in the middle, influencing the metabolism of algae (Becker, 1995). ZANG et al. (201 1) showed that in a microalgae culture, the pH can vary significantly over a day; Wang et al. (2012) showed that high pHs, approximately equal to 1 1, are reached in microalgae cultures without buffering agent in the medium, with the highest values occurring in the exponential phase of growth.

 Regarding all treatments of black water, inoculated and not inoculated with Chlorella vulgaris, it was observed that algae growth was better in the trials in which there was the addition of nutritive salts of BG-1 medium 1. This shows that black water alone does not support algal production, and nutrient addition is required to optimize growth and biomass production.

The results showed that the highest growth rates were obtained in the light water nutrient assays, which are similar to those of microalgae cultivation in conventional culture media. With nutrients, the growth rate of inoculated Chlorophyta microalgae was 0.97 day ^"1 , while without nutrients it was 0.84 day ^{" 1} . This is equivalent to say that the inoculated population made 1, 4 divisions per day in the presence of nutrients, while without nutrients, it took 2 days to double the population (0.57 divisions per day). These results are shown in Figure 6.

 Chlorella vulgaris kept in black water without added nutrients showed

6.23 μg ml_ ^"1 of carbohydrates in the exponential phase, but decreased to 4.34 μg ml_ ^{" 1} at the end of the trials. As expected, black water inoculated with nutrient-enriched Chlorella vulgaris obtained a value of 9.03 μg ml_ ^"1 at the exponential phase and 8.18 μg ml_ ^{" 1} at the end of the experiments, therefore higher than without nutrient addition. These results are shown in Figure 7. Protein concentration results are shown in Figure 8.

 Black water, when kept in light, allowed the growth of native or inoculated microalgae, in the case of Chlorella vulgaris. Growth was higher when black water was enriched with nutrients from culture medium BG-1 1 compared to non nutrient enriched black water.

The description of the wastewater treatment method and the use of the polysaccharide excretory microalgae, object of the present invention, should be considered merely as a possible embodiment, and any particular features introduced therein should be understood solely as something that has been described for ease of understanding. Accordingly, they may not in any way be construed as limiting the invention, which is limited to the scope of the following claims.

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Claims

 1 . RESIDUAL WATER TREATMENT METHOD THROUGH CROP OF POLYESACARIDE EXCRETING MICROALGAS, comprising the following steps:

 (a) collecting in a wastewater treatment tank comprising, in suspension, fine particulates of organic and inorganic material from solid fuels;

 (b) inclusion of nutrients in wastewater;

 (c) inclusion of the polysaccharide excretory microalgae inoculum in the wastewater obtained in step (b);

 (d) separation of the fine particulates by agglutination with the excreted polysaccharides and subsequent decantation of the agglutinated particulates at the bottom of the tank;

 (e) microalgae collection; and

 (f) collection of treated water.

 Method according to claim 1, characterized in that the fine particulates of organic and inorganic material originating from solid fuels suspended in the wastewater are chosen from coal, charcoal, coal or steel grade coke, anthracite, carbon coke. oil or mixtures thereof.

 Method according to Claim 1, characterized in that the polysaccharide excretory microalgae are chosen from the group of Bacillariophyta, Chlorophyta and Cyanophyta.

 Method according to Claim 3, characterized in that the polysaccharide excretory microalgae are chosen from the microalgae of the genera Chlorella, Scenedesmus, Desmodesmus and Monoraphidium; preferably Chlorella vulgaris.

 Method according to Claim 1, characterized in that the nutrients added to the wastewater are chosen from nitrates, phosphates, magnesium, calcium, iron, manganese, copper, molybdenum, cobalt and zinc.

 Method according to Claim 1, characterized in that the microalgae collection step (e) optionally comprises a prior flocculation step followed by the microalgae decantation.

 7. USE OF POLYSACCARIDE EXCRETING MICROALGAS characterized by being for treating waste water comprising, in suspension, fine particulates of organic and inorganic material originating from solid fuels.