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Phytorremediation of urban and fish farming wastewater and growth performance responses of Litopenaeus vannamei using microalgae Chlorella vulgaris

Fitorremediação de efluentes urbanos e piscícolas e respostas do desempenho de crescimento de Litopenaeus vannamei utilizando a microalga Chlorella vulgaris

ABSTRACT

Phytoremediation, as the implementation of microalgae in the treatment of effluents, is used to remove nitrogen compounds and phosphorus that can cause eutrophication in receiving water bodies. The present study evaluated the phytoremediation of urban and fish farming wastewater and growth performance of Litopenaeus vannamei using microalgae Chlorella vulgaris. Microalgae were cultivated stationary with three treatments: T1 sewage treatment station effluent (STS), T2 fish farming effluent (FF) and T3 with standard medium (SM) Guillard f/2), conducted in quintuplicate for each culture medium, using a 20 L carboy for eight days. 5.0 L inoculum (25%) plus 15 L culture medium, T1 STS, T2 FF and T3 SM (75% of each) were used to start the experiment. The shrimp experiment was realized with three treatments in quintuplicates, completely randomized design, during 15 days. The animals were maintained in tanks of 100 L under constantly aerated, resulting in a concentration above 5.0 mg L− 1 of dissolved oxygen, in a density of 70 animals m-3, fed four times per day (8:00, 11:00, 14:00 and 16:00 h) offering to T1 commercial feed (C-Feed) with 35% crude protein, T2 and T3 with commercial feed plus addition of 5.0% dry biomass of C. vulgaris. The best result for algal perfomance, lipid biomass and nutrient removal was with Fish farm wastewater. The additive with the highest performance was FF-Feed. C. vulgaris has a high potential for removing nutrients from wastewater, producing biomass and lipids, in addition to increasing shrimp productivity.

Index terms:
Water quality; nutrients removal; marine shrimp.

RESUMO

Fitorremediação, como implementação de microalgas no tratamento de efluentes, é utilizada para remover compostos nitrogenados e fósforo que podem causar eutrofização. O presente estudo avaliou a fitorremediação de águas residuárias urbanas e piscicultura e o desempenho zootécnico do Litopenaeus vannamei, usando Chlorella vulgaris. As microalgas foram cultivadas em três tratamentos (efluente da estação de tratamento de esgoto T1 STS, efluente aquícola T2 FF e T3 meio padrão (SM) Guillard f/2), conduzidos em quintuplicata para cada meio de cultura, utilizando garrafão de 20 L por 8 dias. 5 L de inóculo (25%) mais 15 L de meio de cultura, T1 STS, T2 FF e T3 SM (75% de cada) foram usados ​​para iniciar o estudo. O experimento com camarões foi realizado com três tratamentos em quintuplicata, em delineamento inteiramente casualizado, durante 15 dias. Os animais foram mantidos em tanques de 100 L sob aeração constante, resultando em concentração acima de 5,0 mg L− 1 de oxigênio dissolvido, densidade de 70 animais m-3, alimentados quatro vezes ao dia (8:00, 11 : 00, 14:00 e 16:00 h) ofertando ração comercial T1 (C-Feed) com 35% de proteína bruta, T2 e T3 com ração comercial mais adição de 5% de biomassa seca de C. vulgaris. O melhor resultado para o desempenho de algas, biomassa lipídica e remoção de nutrientes foi o FF. O aditivo com melhor desempenho foi FF-Feed. C. vulgaris possui alto potencial para remoção de nutrientes de águas residuárias, produção de biomassa e lipídios, além de aumentar a produtividade do camarão.

Termos para indexação:
Qualidade da água; remoção de nutrientes; camarão marinho.

INTRODUCTION

The generation of urban and aquaculture effluents, rich mainly in nitrogen and phosphorus, are challenges faced by society, a large part of aquaculture projects and cities in Brazil do not have an adequate disposal of their effluents before discarding them, which can contaminate and eutrophicate receiving water bodies with excessive growth of macrophytes, cyanophyceae, low specimen diversity, anaerobic conditions and mortality of aquatic organisms (Evans et al., 2017EVANS, L. et al. Effect of organic carbon enrichment on the treatment efficiency of primary settled wastewater by Chlorella vulgaris. Algal Research , 24:368-377, 2017. ). Most studies on effluent treatment are based on physical processes such as stabilization ponds and decanters, chemicals with use flocculation or flotation compounds and biological processes such as water recirculation system that use aggregated biofilters with decantation tanks and processes with and without the presence of oxygen (Arun et al., 2017ARUN, J. et al. Studies on influence of process parameters on hydrothermal catalytic liquefaction of microalgae (Chlorella vulgaris) biomass grown in wastewater. Bioresource Technology, 244:963-968, 2017.). These processes are mainly aimed at removing nitrogenous compounds and phosphorus, resulting in steps with disadvantages due to high infrastructure and professional costs, high operational complexity, demanding large areas, in addition to producing large amounts of sludge (Ajayan; Harilal; Selvaraju, 2018AJAYAN, K. V.; HARILAL, C. C.; SELVARAJU, M. Phycoremediation resultant lipid production and antioxidant changes in green microalgae Chlorella sp. International Journal of Phytoremediation, 20(11):1144-1151, 2018.).

Some bioremoval studies work with plants, such as wetlands that use superior plants and macroalgae (Sujuan et al., 2021SUJUAN, W. et al. The effects of the cooling efficiency of urban wetlands in an inland megacity: A case study of Chengdu, Southwest China. Building and Environment, 204:108128, 2021. ; Saberi et al., 2020SABERI, M. et al. Bioremediation potential o fmacroalgae Gracilaria edulis and Gracilaria changii co-cultured with shrimp wastewater in an outdoor water recirculation system. Environmental Technology & Innovation, 17:100571, 2020.), both processes achieve a lower removal of nitrogen compounds and phosphorus when compared to the use of microalgae, in addition to requiring a longer period for the development of plants and macroalgae. These characteristics make these processes more environmentally and financially costly (Yajing et al., 2021YAJING, C. et al. Review on strategies of close-to-natural wetland restoration and a brief case plan for a typical wetland in northern China. Chemosphere , 285:131534, 2021.). Phytoremediation using microalgae is an excellent alternative for wastewater treatments, as it is more environmentally efficient by absorbing high levels of nutrients, economically with use of biomass and bioactive compounds with high commercial value in various applications in pharmaceutical, cosmetics, biogas, biofuels, biofertilizers, human and animal nutrition industries (Mujtaba; Rizwa; Lee, 2017MUJTABA, G.; RIZWAN, M.; LEE, K. Removal of nutrients and COD from wastewater using symbiotic co-culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris. Journal of Industrial and Engineering Chemistry, 49:145-151, 2017. ; Mujtaba; Lee, 2017MUJTABA, G.; LEE, K. Treatment of real wastewater using co-culture of immobilized Chlorella vulgaris and suspended activated sludge. Water Research, 120:174-184, 2017.). Currently, several studies demonstrate the advances in the efficiency of microalgae in the treatment of urban (Pacheco et al., 2021PACHECO, D. et al. Municipal wastewater: A sustainable source for the green microalgae Chlorella vulgaris biomass production. Applied Science. 11:e2207, 2021. ), industrial (Rajak; Jacob; Kim, 2020RAJAK, R. C.; JACOB, S.; KIM, B. S. A holistic zero waste biorefinery approach for macroalgal biomass utilization: A review. Science Total Environment, 716:e137067, 2020.) and agricultural effluents (Wen et al., 2017WEN, Y. et al. Isolation of an indigenous Chlorella vulgaris from swine wastewater and characterization of its nutrient removal ability in undiluted sewage. Bioresource Technology , 243:247-253, 2017. ), indicating the possible applications of the algal biomass produced, but without using it.

Various effluents can be an excellent alternative for algal cultivation and biomass recovery (Ortenzio et al., 2015ORTENZIO, Y. T. et al. Cultivo de microalgas utilizando resíduos agroindustriais para a produção de biocombustíveis: Perspectivas e desafios. Bioenergia em revista: diálogos, 5:58-65, 2015.), as they are culture media of reduced financial value and with sufficient concentration of nutrients for an efficient growth performance (Wenhai et al., 2019WENHAI, L. et al. Successful blue economy examples with an emphasis on international perspectives. Frontiers in Marine Science, 6:261, 2019.). The removal of nutrients from the culture medium occurs because the microalgae incorporates these to its biomass, effecting a high removal rate (Cao et al., 2018CAO, L. et al. Effect of combining adsorption-stripping treatment with acidification on the growth of Chlorella vulgaris and nutrient removal from swine wastewater. Bioresource Technology , 263:10-16, 2018.) and not causing damage to the ecosystem if chosen correctly, according to environmental factors (Lam et al., 2017LAM, K. M. et al. Cultivation of Chlorella vulgaris using nutrients source from domestic wastewater for biodiesel production: Growth condition and kinetic studies. Renewable Energy , 103:197-207, 2017.).

Phytoremediation, as the implementation of microalgae in the treatment of effluents, is used to remove nitrogen compounds and phosphorus that can cause eutrophication in receiving water bodies (Mohd-Sahib et al., 2017MOHD-SAHIB, A. A. et al. Lipid for biodiesel production from attached growth Chlorella vulgaris biomass cultivating in fluidized bed bioreactor packed with polyurethane foam material. Bioresource Technology , 239:127-136, 2017.). Microalgae can remove more than 95% of nitrogen compounds and 90% of phosphorus in effluents (Praveen; Loh, 2016PRAVEEN, P.; LOH, K. C. Nitrogen and phosphorus removal from tertiary wastewater in an osmotic membrane photobioreactor. Bioresource Technology , 206:180-187, 2016.). The use of native algae, such as Chlorella vulgaris, in the phytoremediation of wastewater is very important because the species presents rapid cell growth, CO2 removal, oxygen production, resistance to contamination, excellent biochemical composition and efficient nutrient absorption (Rodrigues-Sousa et al., 2021RODRIGUES-SOUSA, A. E. et al. Nitrogen supplementation for the production of Chlorella vulgaris biomass in secondary effluent from dairy industry. Biochemical Engineering Journal, 165:e107818, 2021.; Miao et al., 2016MIAO, M. S. et al. Mixotrophic growth and biochemical analysis of Chlorella vulgaris cultivated with synthetic domestic wastewater. International Biodeterioration and Biodegradation, 113:120-125, 2016.; Silva et al., 2015SILVA, J. W. A. et al. Effect of nitrate depletion on lipid accumulation by the marine microalga Nannochloropsis oculata. Boletim do Instituto de Pesca, 41:811-816, 2015.). C. vulgaris was used by Wang et al. (2017WANG, L. et al. Chlorella vulgaris cultivation in sludge extracts from 2,4,6-TCP wastewater treatment for toxicity removal and utilization. Journal of Environmental Management , 187:146-153, 2017.), to evaluate the capacity to remove nitrogen in the form of ammonia or ammonium ion, being able to drastically remove nitrogen compounds in just 48 h, showing its high potential for wastewater treatment. Liu et al. (2017LIU, X. et al. Growth of Chlorella vulgaris and nutrient removal in the wastewater in response to intermittent carbon dioxide. Chemosphere , 186:977-985, 2017. ), which used an urban effluent with carbon dioxide injection and observed a removal of 99% of phosphorus and 87% of ammonia, while Mujtaba and Lee (2017MUJTABA, G.; RIZWAN, M.; LEE, K. Removal of nutrients and COD from wastewater using symbiotic co-culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris. Journal of Industrial and Engineering Chemistry, 49:145-151, 2017. ), used urban treatment wastewater, achieving a removal of 95% ammonia and 90% phosphorus, and Mujtaba, Rizwan and Lee (2017)MUJTABA, G.; RIZWAN, M.; LEE, K. Removal of nutrients and COD from wastewater using symbiotic co-culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris. Journal of Industrial and Engineering Chemistry, 49:145-151, 2017. , found that C. vulgaris removed 70% ammonia and 66% phosphorus when domestic sewage effluent was used.

C. vulgaris is also used as additives in the diet of aquatic organisms, due to its excellent biochemical composition with a high content of proteins, vitamins, minerals and polyunsaturated fatty acids. C. vulgaris biomass improves feed palatability and digestibility, reducing nitrogen and phosphorus content in excreta and optimizing zootechnical performance, achieving environmental and economic gains, promotes good nutrition, elevates individuals immunity and resistance to pathogens and management, allowing for greater cultivation density and greater survival, achieving excellent results in terms of productivity, profitability and sustainability (Alagawany et al., 2021ALAGAWANY, M. et al. Nutritional applications of species of Spirulina and Chlorella in farmed fish: A review. Aquaculture, 542:736841, 2021.). The production of Litopenaeus vannamei is growing rapidly, with a world production of approximately 4.1 million tons (Food and Agriculture Organization of the United Nations - FAO, 2020FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS - FAO. The state of world fisheries and aquaculture: Sustainability in action. 2020. In: Rome, Italy: Fisheries an Aquaculture Technical Paper. 244p.), demanding a food with better nutritional quality, environmentally correct and economically viable. The feed must have a high content of proteins, lipids, vitamins and minerals (Gao et al., 2019GAO, F. et al. Lipid accumulation properties of Chlorella vulgaris and Scenedesmus obliquusi n membrane photobioreactor (MPBR) fed with secondary effluent from municipal wastewater treatment plant. Renewable Energy, 136:671-676, 2019.), important substances for the development of the shrimp and that are part of the composition of C. vulgaris in the necessary amounts.

Silva et al. (2020bSILVA, J. W. A. et al. Influence of microalgae Chlorella vulgaris on zoothenic performance of marine shrimp Litopenaeus vannamei. Brazilian Journal of Development , 6(2):5603-5614, 2020b.), added 0.5% C. vulgaris dry biomass to the feed and compared it with the commercial diet of L. vannamei. The authors found greater final biomass and productivity when adding algal biomass, with an increase of 1.05 times compared to the commercial diet. Pakravan et al. (2017PAKRAVAN, S. et al. Chlorella vulgaris meal improved growth performance, digestive enzyme activities, fatty acid composition and tolerance of hypoxia and ammonia stress in juvenile Pacific white shrimp Litopenaeus vannamei. Aquaculture Nutrition, 24(1):594-604, 2017.), used different experimental diets with C. vulgaris added to L. vannamei feed and confirmed that their biomass can partially or completely replace fishmeal in the feed formulation without causing adverse effects in the zootechnical performance of shrimp, demonstrating environmental feasibility and economic gain when using algae, reducing environmental impacts on fish stocks and feed manufacturing costs due to the high value of fishmeal.

The differential of present study was aggregate environmental and economic value to phytoremediation by using algal biomass as an additive in the L. vannamei feed and enabling the reuse of aquaculture effluent after phytoremediation in the production of fish and shrimp by achieving quality parameters required by cultivated aquatic organisms. This factor demonstrates the importance of multidisciplinarity in the pursuit of sustainable food production and water use. The present work evaluated the C. vulgaris as a phytoremediation of urban and fish farming wastewater and comparing their kinetic yield and water quality, and as an nutritional additive in the feeding of L. vannamei and determining the zootechnical parameters.

MATERIAL AND METHODS

C. vulgaris, Shrimp and Effluents

The microalgae C. vulgaris strain was obtained from the algal culture collection of the Aquaculture Technology Laboratory of the Federal Institute of Ceará (LTA - IFCE) - Aracati Campus, where it is kept in a Guillard f/2 medium (Guillard, 1975GUILLARD, R. R. L. Culture of phytoplankton for feeding marine invertebrates. In: SMITH, W. L.; CHANLEY, M. H. (eds.). Culture of marine invertebrate animal. New York: Plenum Publishing, p.29-60, 1975. ) under controlled temperature conditions (20.0±1.0 ºC) and light intensity maintained at 54 µmol m-2 s-1 using a fluorescent light with a controlled photoperiod (16 h dark:08 h light).

Shrimp were obtained from the shrimp farm CEAQUA, located in the municipality of Beberibe, CE. and transported in a tank of 1,000 L, containing water from the cultivation nursery with salinity 15 and oxygen supply provided by cylinder, distributed by aerotubes with a flux of 6.0 ± 1.0 L air min-1. In LTA - IFCE, shrimp were acclimated and populated in experimental tanks of 100 L with average initial weight of 5.53±0.68 g. The effluents used as a culture medium, urban sewage and fish farming were collected in sewage treatment station (STS) and fish farming (FF) of IFCE - Aracati Campus, respectively. They were transported in two 100 L tanks to the Aquaculture Technology Laboratory (LTA of IFCE - Aracati), during collection, the effluents were filtered in a 25 µm mesh to remove plankton and suspended material, then autoclaved at 120 °C for 15 minutes to eliminate microorganisms that can interfere with algal development and nutrient dynamics.

Experimental design

The inoculum for the beginning of the experiment was produced and acclimated by adding 1,250 mL of C. vulgaris (25%) to 3750 mL of effluents (75%) used as a culture medium, using 5.0 L erlemeyers under the same conditions of temperature, luminosity and aeration of the experiment for three days.

Microalgae were cultivated stationary (Ohse et al., 2009OHSE, S. et al. Produção de biomassa e teores de carbono, hidrogênio, nitrogênio e proteína em microalgas. Ciência Rural, 39(6):1760-1767, 2009. ) with three treatments (T1 effluent from STS, T2 effluent from FF and T3 with standard medium (SM) Guillard f/2), conducted in quintuplicate for each culture medium, using a 20 L carboy for eight days. 5.0 L inoculum (25%) plus 15 L culture medium, T1 STS, T2 FF and T3 SM (75% of each) were used to start the experiment. The microalgae had an initial dry weight of 0.194, 0.196 and 0.191 g L-1 for STS, FF and SM, respectively. After eight days of cultivation, the algal biomass was recovered by chemical flocculation using 2N NaOH, washed to remove the fluctuant and dried in oven with air renewal at 60 °C for 24 h (Silva et al., 2015SILVA, J. W. A. et al. Effect of nitrate depletion on lipid accumulation by the marine microalga Nannochloropsis oculata. Boletim do Instituto de Pesca, 41:811-816, 2015.).

All material used and culture media were sterilized in an autoclave for 15 minutes at 120 °C to avoid any type of contamination. The growing conditions remained constant, with a temperature of 28 ± 1.0 °C, light intensity 108 µmol m-2 s-1, having as light source two 40 W fluorescent lamps, aeration provided by an electromagnetic blower with a flux of 3.0 ± 1.0 L air min-1, pH 7.8 and salinity 0.

The shrimp experiment was realized with three treatments in quintuplicates, completely randomized design, during 15 days. The animals were maintained in tanks of 100 L under constantly aerated by radial blower and aerotubes, resulting in a concentration above 5.0 mg L− 1 of dissolved oxygen, in a density of 70 shrimps m-3, fed four times per day (8:00, 11:00, 14:00 and 16:00 h) offering to T1 commercial feed (C-Feed) with 35% crude protein, T2 and T3 with commercial feed plus addition of 5.0% dry biomass of C. vulgaris, after pretreatment with sonication to break the cell wall, cultivated in effluents of FF-Feed and STS-Feed, respectively (Table 1). The T2 and T3 feed formulation followed the method described by Silva et al. (2020aSILVA, J. W. A. et al. Evaluation of ammonia toxicity in a policulture of Litopenaeus vannamei and Spirulina platensis. Brazilian Journal of Development , 6(2):5603-5614, 2020a.).

Table 1:
Nutritional characteristics of shrimp feed.

The amount of feed started at 5.0% biomass. Animal weight was determined weekly per tank to monitor growth and adjust the amount of feed. 100% of the water in the tanks was renewed daily to maintain the water quality parameters at acceptable levels for the species.

C. vulgaris and shrimp growth performance

Daily, determined the dry biomass weight (DBW) (Lopes et al., 2020LOPES, D. N. M. et al. Dry biomass of diatomacea Chaetoceros gracilis in Different salinities aiming biodiesel production. Brazilian Journal of Development, 6(6):5707-5713, 2020.), to monitor the development of cultures. At the end of the experiment, the kinetic indices were determined: dry biomass yield (DBY) (g), biomass yield (g L−1), biomass productivity (g L-1 day-1), lipid content (%), lipid yield (mg L-1) and lipid productivity (mg L-1 day-1) The weight of the shrimp was monitored weekly to determine the growth of the animals and adjust the amount of feed offered.

The specific growth rate (K) was calculated by the Equation (1) (Ohse et al., 2009OHSE, S. et al. Produção de biomassa e teores de carbono, hidrogênio, nitrogênio e proteína em microalgas. Ciência Rural, 39(6):1760-1767, 2009. ).

K = l n N 2 l n N 2 t 2 t 1 (1)

where N 2 and N 1 are the DBW at time t1 and t2, respectively.

At the end of experiment were calculated, mean final weight (g), daily weight gain (mg day-1), feed conversion rate (FCR), survival (%), final biomass (g), productivity (kg m−3) and bioremediation potential (%).

Determination of lipid content and fatty acid composition in C. Vulgaris

Lipid extraction and quantification of total concentration were evaluated by a modified Bligh and Dyer (1959BLIGH, E. G.; DYER, W. J. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8):911-917, 1959.) method and the characterization of the lipids extracted from C. vulgaris was done by gas chromatography (GC), using the Ce 2-66 method of the AOCS (Association of Official Analytical Chemists - AOCS, 1997ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS - AOCS. Official method Ca 5a-40, Free faty acids. In: FIRESTONE, D. E. (Ed.). Official Methods Recommended Practices. AOCS. Champaign, IL, USA: AOCS Press, p.1-2.1997. ) at the Laboratory of Analysis and Process Development of the Department of Chemical Engineering of the Federal University of Ceará (Fortaleza, Ceará, Brazil).

Water quality

Dissolved oxygen, salinity, pH and temperature were determined (HANNA HI 9829) twice a day (8:00 and 16:00 h) for shrimp cultures. Alkalinity, ammonia, nitrite, nitrate and orthophosphate (American Public Health Association - APHA 2012AMERICAN PUBLIC HEALTH ASSOCIATION - APHA. Standard Methods for the Examination of Water and Wastewater. 22th ed., Washington, DC, USA: APHA Press, 2012. 1496p.) were determined at the beginning and end for algal cultures and weekly for shrimp cultivation. After phytoremediation of urban and aquaculture effluents, nutrient concentrations were compared to CONAMA Resolution n°. 357/2005 (Brasil, 2005) and Boyd and Tucker (2014BOYD, C. E.; TUCKER, C. S. Handbook for aquaculture water quality. Craftmaster, Auburn, USA: Springer, 2014. 439p.).

Statistical analysis

Analysis of variance (ANOVA) (P ≤ 0.05) was used for analyzed water quality, C. vulgaris and shrimp performance. When significant differences were found between treatments (P ≤ 0.05) used Tukey’s test. All statistical data were performed using Assistat 7.6.

RESULTS AND DISCUSSION

Microalgae growth

The three tests showed similar behavior and satisfactory growth for the different culture medium, but with different productivity. In the first two days, the cells adapting to the new culture conditions. After acclimatization, the cells showed exponential growth due to the favorable conditions of light and nutrient, with highlighted to FF that got better development, followed by SM and STS, respectively, until the final in the stationary phase of cultivation with reduced growth rates (Figure 1).

Figure 1:
Growth kinetics (g L-1) of three tested C. vulgaris under different culture medium. (FF) Fish farming effluent, (STS) Sewage treatment station and (SM) Standard medium.

According to Silva et al. (2020aSILVA, J. W. A. et al. Evaluation of ammonia toxicity in a policulture of Litopenaeus vannamei and Spirulina platensis. Brazilian Journal of Development , 6(2):5603-5614, 2020a.), after inoculation of a microalgae in a nutrient-enriched culture medium, population grows over time, generally presents a curve with five distinct phases. This nutrient absorption capacity demonstrates the efficiency of C. vulgaris in the treatment of effluents, being important in the management of water resources, as the effluent discarded will respect environmental standards, reducing costs and the load of toxic and pathogenic nutrients in the environment.

In all tests, the growth rate had an initial tendency to increase until the third day of cultivation, with a tendency to decrease until the end of the experiment on the eighth day (Figure 1). Similar to the present experiment, studies such as of Katiyar et al. (2021KATIYAR, R. et al. An integrated approach for phycoremediation of municipal wastewater and production of sustainable transportation fuel using oleaginous Chlorella sp. Journal of Water Process Engineering, 42:102183, 2021.), Zhang, Ren and Jiang (2021ZHANG, C.; REN, H. X.; JIANG, L. Cultivation of Chlorella protothecoides in polyglutamic acid wastewater for cost-effective biodiesel production. Arabian Journal of Chemistry, 14:103108-103117, 2021.), Wu et al. (2020WU, J. Y. et al. Immobilized Chlorella species mixotrophic cultivation at various textile wastewater concentrations. Journal of Water Process Engineering , 38:101609, 2020.), report, in Chlorella sp. cultures, the daily deceleration of growth rate as the nutrients are absorbed, mainly nitrogen and phosphorus, which are essential nutrients for algae growth.

The highest specific growth rates of C. vulgaris, during the experiment, were achieved in FF, followed by SM and STS. There was a significant difference during the growing period between FF, STS and SM, with FF with an increase in growth rate 1.23 and 1.85 times greater than STS and SM, respectively. The growth of STS compared to SM was significantly slower, with the SM culture 1.13 times fastest than STS (Table 2). Lower concentrations of nitrogen compounds, possibly, influenced a higher growth rate, as high absorbed concentrations can be toxic to C. vulgaris.

Table 2:
Means and standard deviation of specific growth rate (K d-1) of C. vulgaris in different culture medium.

Kong et al. (2021KONG, W. et al. Chlorella vulgaris cultivation in simulated wastewater for the biomass production, nutrients removal and CO2 fixation simultaneously. Journal of Environmental Management, 284:112070, 2021.), used simulated wastewater aiming to produce biomass of C. vulgaris, their results presented that maximal specific growth rate of C. vulgaris was 0.21-0.35 d-1 and 0.33-0.43 d-1 at 90 mg L-1 and 60 mg L-1 of nitrogen source, respectively. The authors indicate in this study that a higher concentration of nitrogen source reduces the specific growth rate, especially in the first days, as algal cells are adapting to new culture conditions and possible toxicity due to excess nutrient.

Biomass and lipid yield of C. Vulgaris

Table 3 demonstrates that the FF and SM treatments were similar to each other and different with STS, showed higher dry biomass, biomass yield and biomass productivity, possibly, because FF and SM absorb phosphorus more efficiently, with 100% removal on both, than STS (69.7%). As phosphorus was fully absorbed from the culture environment in FF and SM, the cells did not accumulate this nutrient, reducing storage and increasing utilization, resulting in greater vegetative growth in 8 days of culture. Dry biomass, biomass yield and biomass productivity of FF and SM grew up 1.35 e 1.22 times more, respectively, than STS.

Table 3:
Means and standard deviation of kinetic algal and lipid yield in C. vulgaris cultures with fish farm (FF), sewage treatment station (STS) effluents and standard medium (SM), during eight days in 20 L of cultivation.

High concentrations of nitrogen and phosphorus reduce lipid synthesis, because they offer favorable cultivation conditions, not inducing stress on the cells and energy reserve in the form of lipids, which reduces the production of bioactive compounds with high economic and nutraceutical value.

Carneiro et al. (2021CARNEIRO, M. et al. Growth and bioactivity of two chlorophyte (Chlorella and Scenedesmus) strains co-cultured outdoors in two different thin-layer units using municipal wastewater as a nutrient source. Algal Research, 56:102299, 2021.), evaluated the growth of Chlorella using municipal wastewater as a nutrient source. Their results showed that the co-cultures grew well in the centrate, achieving the maximum biomass densities 1.3 g DW L-1 by the end of semi-continuous regime in thin-layer cascade, which increases the algal photosynthetic area, resulting in more chemical energy and cell division, on the other hand, raises production costs. In the present work, the economic costs are reduced, as it did not use laminar culture mechanisms or bioreactors, but achieving good algal and lipid productivity results. Molazadeh et al. (2019MOLAZADEH, M. et al. Influence of CO2 concentration and N: P ratioon Chlorella vulgaris assisted nutrient bioremediation, CO2 biofixation and biomass production in a lagoon treatment plant. Journal of the Taiwan Institute of Chemical Engineers, 96:114-120, 2019.) cultivated C. vulgaris in urban sewage treatment plant effluent, generating maximum biomass concentration, biomass productivity of 0.7900 g L−1 and 0.0850 g L−1 d−1, respectively. Even using CO2, the authors found inferior result of biomass concentration and biomass productivity when compared to the three tests of the cultivation of C. vulgaris in the present study, possibly due to the inadequate N: P ratio for biomass production.

The FF treatment had the best lipid results along with SM in the lipid content and lipid productivity. The STS got the lowest lipid yield of the three treatments, followed by SM second minor lipid yield and FF showing the best result (Table 2). The cultures FF and SM accumulated more lipid content, they were improved by 1.59 and 1.35 times in comparison with SM. The lipid productivity of FF and SM were 2.16 and 1.65 times higher, respectively, than STS, possibly, because of phosphorus depletion in cultivation and energy accumulation in the form of lipids.

Katiyar et al. (2021KATIYAR, R. et al. An integrated approach for phycoremediation of municipal wastewater and production of sustainable transportation fuel using oleaginous Chlorella sp. Journal of Water Process Engineering, 42:102183, 2021.), produced some species of Chlorella. Their lipid productivity of Chlorella minutissima, C. sorokiniana, was 36.66 and 48.33 mg L-1 day-1, respectively, in municipal wastewater. Amini, Wang and Shahbazi (2016AMINI, H.; WANG, L.; SHAHBAZI, A. Effects of harvesting cell density, medium depth and environmental factors on biomass and lipid productivities of Chlorella vulgaris grown in swine wastewater. Chemical Engineering Science, 152:403-412, 2016.) evaluated the lipid productivity of C. vulgaris cultivated in swine wastewater, obtaining the highest lipid productivity 40.41 mg L-1 day-1. Gao et al. (2019GAO, F. et al. Lipid accumulation properties of Chlorella vulgaris and Scenedesmus obliquusi n membrane photobioreactor (MPBR) fed with secondary effluent from municipal wastewater treatment plant. Renewable Energy, 136:671-676, 2019.), used secondary effluent from municipal wastewater treatment plant to cultived C. vulgaris, their lipid content and lipid productivity were 13% and 25.76 mg L-1 day-1, respectively. The three studies cited obtained superior results to the present study, possibly due to the stress of nutrient reduction, stimulating lipid biosynthesis in the cells of the C. vulgaris in their respective studies. Future studies are needed with the dilution of fish farm effluents to reduce the concentration of nitrogen compounds and induce lipid increase.

Characterization of the fatty acids present in C. vulgaris

As show in Table 4, the characterization of fatty acids from C. vulgaris identified a significant difference between the effluents used (FF and STS), but with similar sums of SFA and MUFA. The MUFAs showed proportions greater than 63% of the total fatty acids methyl ester (FAME) for FF and STS, with a significant increase in C16:1 and C22:1 and a reduction in C18:1 when using FF as the culture medium.

Table 4:
Percentage of fatty acids in oil obtained from C. vulgaris cultures with fish farm (FF), sewage treatment station (STS) effluents and standard medium (SM).

The SM obtained a considerably higher C16:0 concentration (5.04-3.51 times), resulting in a higher SFA concentration than the other treatments. Concentrations of C18:2 were similar for FF and SM, while the proportion for STS significantly increased by 27.1%. There was an increase in C18:0 of ​​57.4% in FF and 31.16% in STS compared to SM. The use of FF and STS effluents increased the biosynthesis of C18:0, C16:1, C18:1 and C22:1 fatty acids, while significantly reducing the synthesis of C16:0, when compared to SM. The results demonstrate that C. vulgaris cultivated in aquaculture and urban effluents produced a higher concentration of MUFAs and PUFAs, respectively, unsaturated fatty acids related to improved immunity (Guo et al., 2021GUO, F. et al. The association between fatty acid intake and breast cancer based on the NHANES and Mendelian randomization study. Cancer Epidemiology, 73:101966, 2021.), increased brain functions (Cisbani et al., 2021CISBANI, G. et al. Murine and human microglial cells are relatively enriched with eicosapentaenoic acid compared to the whole brain. Neurochemistry International, 150:105154, 2021.) and nervous system (Gu et al., 2021GU, M. et al. Endogenous ω-3 fatty acids in Fat-1 mice attenuated depression-like behaviors, spatial memory impairment and relevant changes induced by olfactory bulbectomy. Prostaglandins, Leukotrienes and Essential Fatty Acids, 171:102313, 2021.), reduced risk of cardiovascular diseases ( Evangelista-Silva et al., 2021EVANGELISTA-SILVA, P. H. et al. Intestinal GLUT5 and FAT/CD36 transporters and blood glucose are reduced by a carotenoid/MUFA-rich oil in high-fat fed mice. Life Sciences, 279:119672, 2021.; Mezouar et al., 2016MEZOUAR, D. et al. In vitro effects of vitamins C and E, n-3 and n-6 PUFA and n-9 MUFA on placental cell function and redox status in type 1 diabetic pregnant women. Placenta, 42:114-121, 2016.). They are also used in the formulation of drugs such as anti-inflammatory and antioxidants (Khaddaj; Morin; Rousseau, 2016KHADDAJ-MALLAT, R.; MORIN, C.; ROUSSEAU, E. E. Novel n-3 PUFA monoacylglycerides of pharmacological and medicinal interest: Anti-inflammatory and anti-proliferative effects. European Journal of Pharmacology, 792:70-77, 2016.; Marrapu et al., 2020MARRAPU, B. et al. Chemo-enzymatic synthesis, characterization, in vitro antioxidant capacity and oxidative stability studies of novel phosphatidylcholines withω-3/ω-6PUFAs and phenolic acids. Food Research International, 131:109010, 2020.), in food industries as a human/animal food supplement (Rojas et al., 2020ROJAS, V. M. et al. Whey protein supplement as a source of microencapsulated PUFA-rich vegetable oils. Food Bioscience, 37:100690, 2020.; Hossain; Peng; Samll, 2021HOSSAIN, M. S.; PENG, M.; SAMLL, B. C. Optimizing the fatty acid profile of novel terrestrial oil blends in low fishmeal diets of rainbow trout (Oncorhynchus mykiss) yields comparable fish growth, total fillet n-3 LC-PUFA content, and health performance relative to fish oil. Aquaculture , 545:37230, 2021.) and cosmetics sector such as moisturizers and lubricants (Yarkent; Gurlek; Oncel, 2020YARKENT, Ç.; GURLEK, C.; ONCEL, S. S. Potential of microalgal compounds in trending natural cosmetics: A review. Sustainable Chemistry and Pharmacy, 17:100304, 2020.). The SFAs obtained from C. vulgaris biomass are important in the manufacture of waxes and oil for burning and energy production (Nirmala; Dawn, 2021NIRMALA, N.; DAWN, S. S. Optimization of Chlorella variabilis. MK039712.1 lipid transesterification using response surface methodology and analytical characterization of biodiesel. Renewable Energy , 179:1663-1673, 2021.; Davoodbasha et al., 2021DAVOODBASHA, M. et al. Biodiesel production through transesterification of Chlorella vulgaris: Synthesis and characterization of CaO nanocatalyst. Fuel, 300:121018, 2021.).

Fazal et al. (2021FAZAL, T. et al. Integrating bioremediation of textile wastewater with biodiesel production using microalgae (Chlorella vulgaris). Chemosphere, 281:130758, 2021. ), evaluated the lipid production of C. vulgaris cultivated in textile wastewater, found the SFAs yield in diluted medium higher (11.07 mg g-1) than the undiluted medium (9.12 mg g-1), mainly C16:0 (10.64 mg g-1), C16:0 concentration similar to the present study when using effluents as a culture medium. However, when using undiluted medium, the study obtained the highest concentration of MUFAs, with emphasis on C22:1 (3.49 mg g-1), concentration significantly lower than that of the present study. The best result for PUFAs was achieved when using standard medium, with 31.08 mg g-1 to C18:2, indicating that textile wastewater is more efficient in the synthesis of C18:2 than the effluents used in present study. Zheng et al. (2021ZHENG, S. et al. Bioremediation of Pyropia-processing wastewater coupled with lipid production using Chlorella sp. Bioresource Technology , 321:124428, 2021.), used Pyropia-processing wastewater as a culture medium to Chlorella sp. The Pyropia-processing wastewater was more efficient in the biosynthesis of SFAs and PUFAs, mainly C16:0 (22.28%) and C18:2 (17.69%), respectively, however, it synthesized less MUFAs than the effluents used in the present study. The synthesis of fatty acids depends on substrates and enzymes, can produce different SFA, MUFA and PUFA at different concentrations, expanding their use in diverse applications.

Nutrient removal

Table 5 shows that the reductions in inorganic nutrients from effluents and standard culture medium were significant when using C. vulgaris as a phytoremediator. The cultivations of C. vulgaris in FF and STS were more efficient in reducing the ammonia concentration, with 80.7 and 75.7% removal, respectively. Nitrite decreased in its entirety for the three treatments, however, nitrate had similar removal, with 95.1, 92.0 and 98.6% for FF, STS and SM, respectively. Cultures with FF and SM totally absorbed phosphorus, this efficiency in phosphorus consumption is of great interest for the treatment of aquaculture effluents and the feasibility of water reuse. Unlike STS, which absorbed 69.7%, showing a significantly lower efficiency.

Table 5:
Mean and standard deviation of nutrient removal in in C. vulgaris cultures with fish farm (FF), sewage treatment station (STS) effluents and standard medium (SM).

Microalgal growth depends mainly on three factors: light, temperature and amount of nutrients available. When considering a medium that is not nutrient-limited, the factors that interfere with algal growth are light and temperature. And the light will still depend on the photoperiod that directly influences photosynthesis, cell division and growth (Amini; Wang; Shahbazi, 2016AMINI, H.; WANG, L.; SHAHBAZI, A. Effects of harvesting cell density, medium depth and environmental factors on biomass and lipid productivities of Chlorella vulgaris grown in swine wastewater. Chemical Engineering Science, 152:403-412, 2016.). According to Fernández-Linares et al. (2017FERNÁNDEZ-LINARES, L. C. et al. Assessment of Chlorella vulgaris and indigenous microalgae biomass with treated wastewater as growth culture medium. Bioresource Technology , 244:400-406, 2017. ), even considering the same species of microalga under the same growing conditions, its growth may be variable. With this, the factors that determine the growth were all controlled during the cultivation, revealing an efficient cell multiplication, since it was provided light, temperature, and nutrients necessary for algal development. Znad et al. (2018ZNAD, H. et al. Bioremediation and nutrient removal from wastewater by Chlorella vulgaris. Ecological Engineering, 110:1-7, 2018.) used C. vulgaris and obtained a result compared to FF, with 79% removal of total nitrogen and 100% removal of total phosphorus from effluents from wastewater treatment plants, and with alternative medium, in this same study, total phosphorus removed was 100% and for alternative medium and 79.4% total nitrogen removal rate was achieved using alternative medium as culture medium. The cultivation of microalgae reduced by up to 90.51% of total nitrogen and 91.54% of total phosphorus, improving the quality of swine wastewater, converting these nutrients into biomass that can be used in the production of biogas or syngas, and reducing possible eutrophication in the receiving bodies of this final effluent (Wen et al., 2017WEN, Y. et al. Isolation of an indigenous Chlorella vulgaris from swine wastewater and characterization of its nutrient removal ability in undiluted sewage. Bioresource Technology , 243:247-253, 2017. ). These studies point to microalgae as an alternative that added value and a high nutrient removal rate, mainly of the Chlorella genus, which absorb the compounds under conditions of high nitrogen and phosphorus load, converting them into biomass rich in proteins, fatty acids, minerals and vitamins, in addition to producing biogas as an energy source.

Ayatollahi, Esmaeilzadeh and Mowla (2021AYATOLLAHI, S. C.; ESMAEILZADEH, F.; MOWLA, D. Integrated CO2 capture, nutrients removal and biodiesel production using Chlorella vulgaris. Journal of Environmental Chemical Engineering. 9:104763, 2021.) cultured C. vulgaris municipal wastewater integrated with CO2 capture and obtained ammonia, nitrate and phosphorus removal of 99, 81.05 and 87.95%, respectively, demonstrating that C. vulgaris is efficient in removing nitrogen compounds and phosphorus, essential compounds for amino acid biosynthesis, cell membrane formation and cellular energy production. This result was similar to the present study, with capacity total nitrogen removal exceeding 80% and almost 70% phosphorus removal in effluents. Praveen and Loh (2016PRAVEEN, P.; LOH, K. C. Nitrogen and phosphorus removal from tertiary wastewater in an osmotic membrane photobioreactor. Bioresource Technology , 206:180-187, 2016.) used effluent from the tertiary wastewater and reported a high efficiency of C. vulgaris in the removal efficiency for ammonia, nitrate and phosphosrus reached as high as 95%, 53% and 89%, respectively. Different of present study, C. vulgaris prefered ammonia as a primary source of nitrogen, revealing that there are several factors that influence the growth and consequently the removal rate, since the microalgae incorporate nitrogen into the biomass for protein biosynthesis.

Shrimp zootechnical parameters

Regarding shrimp growth performance, significant differences was found in final biomass and yield with the following result among treatments: FF-Feed > C-Feed > STS-Feed (Table 6). Final biomass and yield of FF-Feed were 1.05 and 1.13 times greater than C-Feed and STS-Feed, respectively, C-Feed was 1.07 times greater than STS-Feed. Survival, even without significant difference, influenced the differences in final biomass and yield, as it increased these data at the end of the experiment. For the other parameters, no significant differences were found. The zootechnical indices in FF were better, possibly due to the higher lipid concentration of the biomass that influenced the shrimp immunity, higher average weight and final biomass obtaining better productivity and profitability.

Table 6:
Means and standard deviation of zootechnical parameters of L. vannamei marine shrimp after inclusion of C. vulgaris in the diet.

Silva et al. (2020bSILVA, J. W. A. et al. Influence of microalgae Chlorella vulgaris on zoothenic performance of marine shrimp Litopenaeus vannamei. Brazilian Journal of Development , 6(2):5603-5614, 2020b.), added 0.5% dry biomass of C. vulgaris to the feed and compared it with the commercial diet of L. vannamei. Similar to the present study, the authors found greater final biomass and productivity when adding algal biomass, with an increase of 1.05 times compared to the commercial diet. These results indicate that the addition of C. vulgaris to L. vannamei feed increases the shrimp farming’s zootechnical, health and economic indices, in addition to increasing the digestibility of the feed and reducing the concentration of toxic nutrients in the environment. Silva et al. (2021)SILVA, A. E. M. et al. Effect of Brachionus plicatilis and Navicula sp. on Pacific white shrimp growth performance, Vibrio, immunological responses and resistance to white spot virus (WSSV) in nursery biofloc system. Aquaculture , 535:736335, 2021., cultivated marine shrimp in two systems, one with bioflocs and the other with bioflocs plus microalgae Navicula sp. The microalgae system obtained final weight and yield 1.23 and 1.22, respectively, superior to the system without microalgae, demonstrating the efficiency of adding microalgae to the marine shrimp diet in terms of production and productivity gains, in addition to increasing shrimp immunity due to bioactive compounds present in the alga.

Ge et al. (2016GE, H. et al. Effect of microalgae with semicontinuous harvesting on water quality and zootechnical performance of white shrimp reared in the zero water exchange system. Aquacultural Engineering, 72-73:70-76, 2016.), evaluated the effects of feed microalgae in L. vannamei culture. They compared the zootechnical indices when cultivating the shrimp in tanks without microalgae and tanks with C. vulgaris. Due to the absorption of C. vulgaris microalgae cells by shrimp in the microalgae tank, final weight and productivity were significantly larger (17.64 g and 4.20 kg m-3, respectively) that the results of the tank without microalgae (14.30 g and 2.41 kg m-3, respectively), corroborating the present study on the efficiency of C. vulgaris in increasing biomass and productivity in marine shrimp farming.

Water quality

Table 7 demonstrates the concentration of water quality variables in the treatments were within the ranges recommended for the marine shrimp farming (Boyd and Tucker, 2014BOYD, C. E.; TUCKER, C. S. Handbook for aquaculture water quality. Craftmaster, Auburn, USA: Springer, 2014. 439p.).

Table 7:
Means and standard deviations of water quality parameters of the marine shrimp L. vannamei culture during the experiment with addition of C. vulgaris in the diet.

Based on Boyd and Tucker (2014BOYD, C. E.; TUCKER, C. S. Handbook for aquaculture water quality. Craftmaster, Auburn, USA: Springer, 2014. 439p.) and CONAMA Resolution n°. 357/2005 (Brasil, 2005BRASIL. Conselho Nacional do Meio Ambiente. Resolução n° 375, de 17 de março de 2005. Brasília, DF, Brasil: Diário Oficial da União, Seção 1, p.58-63, 2005.), after phytoremediation with C. vulgaris, FF and SM showed totally efficient removal of water quality variables, enabling the reuse and discharged in accordance with current legislation, nitrogen is a limiting factor for eutrophication for freshwater classes 1 and 2, not exceeding the total nitrogen value of 1.27 mg L-1 for lentic environments and 2.18 mg L-1 for lotic environments, at the reference flow rate. Phosphorus concentrations for FF and SM, with total removal, are in accordance with current legislation for disposal of lotic, intermediate and lentic environments of class 1 for fresh water, as the maximum requirement is 0.020 mg L-1. In contrast, STS was not efficient in the removal of ammonia and phosphorus, above recommended for the correct disposal of effluents (Brasil, 2005BRASIL. Conselho Nacional do Meio Ambiente. Resolução n° 375, de 17 de março de 2005. Brasília, DF, Brasil: Diário Oficial da União, Seção 1, p.58-63, 2005.), being indicated a longer phytoremediation time for a possible reduction of these parameters (Table 8). Silva et al. (2020aSILVA, J. W. A. et al. Evaluation of ammonia toxicity in a policulture of Litopenaeus vannamei and Spirulina platensis. Brazilian Journal of Development , 6(2):5603-5614, 2020a.), Ge et al. (2016GE, H. et al. Effect of microalgae with semicontinuous harvesting on water quality and zootechnical performance of white shrimp reared in the zero water exchange system. Aquacultural Engineering, 72-73:70-76, 2016.) and Maliwat et al. (2021MALIWAT, G. C. F. et al. Growth and immune response of pond-reared giant freshwater prawn Macrobrachium rosenbergii post larvae fed diets containing Chlorella vulgaris. Aquaculture and Fisheries, 6(5):465-470, 2021. ), demonstrate the positive effects on water quality and shrimp performance when using C. vulgaris, in water resources management, profitability and public health.

Table 8:
Means and standard deviation of water quality parameters after biorremediation with C. vulgaris.

The study demonstrates the importance of advancing the verticalization of water use, with use in the cultivation of aquatic organisms, biotreatment, reuse and production of algal biomass for shrimp feed additive, adding value to the product, reducing environmental impacts and generating employment in the sector. These results demonstrate promising economic, social and environmental benefits of the production of microalgae for the extraction of proteins, lipids, vitamins, carotenoids and polysaccharides of high financial value and used in the animal and human nutrition, nutraceutical, cosmetic, biofertilizer and aquatic biotreatment industries. More studies should be developed with the addition of bioactive compounds from microalgae in the diet, for a better understanding of the zootechnical parameters and immunity of the shrimp.

CONCLUSIONS

C. vulgaris proved to be an excellent alternative to treatment of farming fish and urban wastewater, efficiently reducing nutrients, increasing water quality and enabling reuse, mitigating environmental problems such as eutrophication, in addition to effective productivity of algal biomass and benefits for the algaculture, aquaculture nutrition, wastewater treatment and water reuse sectors. The addition of C. vulgaris to marine shrimp feed, influenced the quantity and lipid quality of diet, with emphasis to FF-feed, which presented better zootechnical parameters, increasing productivity and profitability.

AUTHOR CONTRIBUTION

Conceptual Idea: Lopes, D. N. M.; Silva, J. W. A.; Methodology design: Lopes, D. N. M., Silva, A. C. T.; Oliveira, I. B. R.; Fernandes, F. A. N.; Silva, J. W. A.; Data collection: Lopes, D. N. M.; Silva, A. C. T.; Santos, S. F. M.; Data analysis and interpretation: Lopes, D. N. M.; Oliveira, I. B. R.; Santos, S. F. M.; Fernandes, F. A. N. and Writing and editing: Lopes, D. N. M.; Silva, J. W. A.

ACKNOWLEDGMENTS

To the financing of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES.

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Publication Dates

  • Publication in this collection
    24 Nov 2021
  • Date of issue
    2021

History

  • Received
    10 Aug 2021
  • Accepted
    15 Oct 2021
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