Assessment of the potential for treating biodiesel wash water with monocultures and co-cultures between the microalgae Chlorella sp. and the cyanobacterium Oscillatoria sp.

Como Citar:

GUERRA, Witter Duarte; CHIESA, Jaqueline Elise Garcia; ALBUQUERQUE, Karolynne Gomes; MARTINS, Alexandre de Matos; ROCHA, Lucas Matheus da; FARIA, Anizio Marcio de; et al. Assessment of the potential for treating biodiesel wash water with monocultures and co-cultures between the microalgae Chlorella sp. and the cyanobacterium Oscillatoria sp. Revista Sociedade Científica, vol. 8, n. 1, p. 1817-1837, 2025. https://doi.org/10.61411/rsc202594618

DOI: 10.61411/rsc202594618

Knowledge area:

Biotechnology

Sub-area:

Biofuels; Microbiology; Phycology

Keywords: Microalgae; Cyanobacteria; Biofuel; Cultivation.

Published: 23 de setembro de 2025.

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Abstract

Microalgae and cyanobacteria, due to their high potential for lipid accumulation, are considered promising for biodiesel production. The joint cultivation of these microorganisms aims to optimize the use of nutrients and stimulate growth. However, biological, physical and chemical factors affect metabolism and nutrient storage, requiring improvements in cultivation methods. This study proposes the separate and joint cultivation of microalgae and cyanobacteria, using biodiesel effluent, such as washing water, as a culture medium. Despite this, preliminary results indicate low effectiveness due to sensitivity to contaminants present in the effluent. It is necessary to develop new methodologies to achieve better results.

Avaliação do potencial de tratamento de água de lavagem de biodiesel com monoculturas e coculturas entre a microalga Chlorella sp. e a cianobactéria Oscillatoria sp.

Resumo

Microalgas e cianobactérias, devido ao seu alto potencial de acumulação de lipídios, são consideradas promissoras para a produção de biodiesel. O cultivo conjunto desses microrganismos visa otimizar o uso de nutrientes e estimular o crescimento. Porém, fatores biológicos, físicos e químicos afetam o metabolismo e armazenamento de nutrientes, exigindo melhorias nos métodos de cultivo. Este estudo propõe o cultivo separado e conjunto de microalgas e cianobactérias, utilizando efluente de biodiesel, como a água de lavagem, como meio de cultura. Apesar disso, resultados preliminares indicam baixa eficácia devido à sensibilidade aos contaminantes presentes no efluente. É necessário desenvolver novas metodologias para alcançar melhores resultados.

Palavras-chave: Microalgas; Cianobactérias; Biodiesel; Água; Cultivo.

• • Introduction

Energy can come from renewable or non-renewable sources, and non-renewable energy sources are considered limited and exhaustible as their regeneration in nature takes a long time. Non-renewable sources include fossil fuels (oil, coal and natural gas) and energy from nuclear fission. Renewable energy sources are not exhaustible and are available in nature for a long period. Renewable sources include solar energy, wind energy, hydropower and biomass, among others [1.].

Research states that of the various alternative sources, the cultivation of algae for the production of biofuels has received increasing attention. Through photosynthesis, microalgae can use water, CO2 and sunlight to synthesize biomass, feedstock for the production of biofuels (e.g. biodiesel, bioethanol and biogas) and high-value biological products such as cosmetics, fine chemicals and different types of acids [2.].

Microalgae have been used all over the world for different purposes. In addition to the high content of lipids and carbohydrates, they can be considered an excellent raw material due to their rapid growth and high efficiency in CO2 fixation. They can be used to produce biofuels, obtain pigments, treat industrial effluents and sewage [3.].

In addition to all these applications in industry, microalgae have gained great space when it comes to bioenergy, they are considered third generation biofuels, as they have great potential for the production of many biofuels such as biodiesel, bioethanol and biohydrogen. The use of microalgae is seen as a viable source of biomass and has caused great expectations in the energy sector [4.].

With this surplus in mind, the present study aims to cultivate a species of microalgae, Chlorella sp., and a species of cyanobacteria Oscillatoria sp. in both monoculture and co-culture systems, aiming at lipid and biomass production using effluent from the biodiesel production cycle (biodiesel wash water). This would be one of the alternatives to reduce dependence on fossil fuels, utilizing the oils from microalgae and cyanobacteria, along with their biomass, for various purposes.

• • Methodology

The analyses of the samples under microscopy were carried out at the Microscopy, Morphometry and Identification Laboratory (LAMMI), a laboratory at CT-Infra 3 of the Biological Sciences Course at the Pontal Institute of Exact and Natural Sciences, Federal University of Uberlândia – UFU, Pontal Campus in Ituiutaba – MG. 

All images were obtained by the Leica stereomicroscope model S8 APO, with digital image capture and by the Nikon Ecplipse E200 Microscope.

The images obtained by the stereomicroscope and the microscope were used for different purposes, since the culture samples are very small in size, requiring the adoption of tools for magnification and verification of aspects such as: contamination of microalgae cultures with cyanobacteria.

For the production of biomass, lipids and biodiesel from microalgae and cyanobacteria, the microalgae Chlorella sp. and a species of cyanobacteria Oscillatoria sp. were used in a co-culture system. The tests were carried out in the laboratories of CT-Infra 2 and 3 of the Biological Sciences and Chemistry Courses linked to the Institute of Exact and Natural Sciences of Pontal, Federal University of Uberlândia - UFU, Pontal Campus in Ituiutaba - MG.

 The microalgae and cyanobacteria strains used in this work were kindly provided by the Postgraduate Program in Biofuels of the Federal University of Jequitinhonha and Mucuri Valleys – UFVJM, Diamantina – MG.

 The strains were isolated at the Laboratory of Biofuels and Green Chemistry (LABIOGREENC) and maintained at the Laboratory of Microscopy, Morphometry and Identification (LAMMI). The experimental tests were performed at the Separation and Chromatography Materials Research Laboratories and at the Materials Chemistry Laboratory. All tests were performed in controlled environments.

Based on cross-contamination of microalgae (Chlorella sp.) and cyanobacteria (Oscillatoria sp.), preliminary tests were carried out to verify the potential and profile of this new culture/co-culture. It is known that there are ecological interactions in natural environments between algae-algae, algae-cyanobacteria and cyanobacteria-cyanobacteria that can have harmonious or even disharmonious ecological relationships. These tests seek to understand these ecological relationships and verify the potential of co-culture in the area of biofuels.

For the tests performed with the co-culture (microalgae-cyanobacteria), the Chu medium was used without the addition of vitamins and without the Ph correction [5.].

To cultivate microalgae, Chu medium was used, with 5 mL of medium for 800 mL of distilled water and 200 mL of inoculum (co-culture). The photobioreactors used for the tests were, according to some researchers, who in their studies used horizontal tray-type reactors, coated with PVC plastic to prevent water evaporation [4.].

The photobioreactors were arranged on 3 shelves uniformly illuminated with two “daylight” lamps with a 12/12h photoperiod.

In order to compare cell growth, biomass productivity and lipid yield, the co-cultivation was subjected to the batch regime, where the experiments were carried out in a laboratory environment with controlled light, nutrients and aeration, thus reducing the risk of new contamination.

The inoculum age was defined with a 21-day cultivation, according to research related to microalgae, which presented in their works cultivations of microalgae Scenedesmus sp. and Chlorella sp. and obtained the best results in relation to biomass-oil and lipid yield with the inoculum age of 21 days [6.,7.].

To determine cell growth, the spectrophotometry method in the visible region was adopted, to monitor biomass production from growth in relation to cultivation time, with absorbance measurements at a wavelength of 570 nm [5.].

To determine the possibility of microalgae and cyanobacteria being potential bioremediation agents in different environments, tests were carried out to verify whether the co-culture system in the microalgae-cyanobacteria relationship is capable of surviving and treating the effluent in question, and subsequently verify what quantity of effluent this system has the capacity to absorb from these residues.

The effluent was kindly provided by the Science and Technology Program – Chemistry, of the Federal University of ABC – UFABC, Santo André – SP. The effluent in question was divided into three different types of effluents:

• Effluent A: biodiesel wash water (Methanol);

• Effluent B: biodiesel wash water (Ethanol);

• Effluent C: biodiesel wash water (Ethanol + Methanol), as shown in Figure 1.

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Figure 1: Biodiesel wash water (ethanol and methanol).

Source: Authors (2024).

Various concentrations of biodiesel wash water were evaluated in order to examine the capacity of this new co-culture as a bioremediation agent in biodiesel wash water.

During the experiments carried out with biodiesel wash water, the concentrations of each effluent (A, B and C) were measured in order to investigate whether changes in the concentrations of these effluents could lead to improvements in the corresponding treatments [4.,6.].

Twenty-four experiments were conducted, in which the effluent concentrations were varied under four different conditions: no addition of the specific effluent, half the concentration, standard concentration and double the concentration as indicated in Table 1. These variations were applied both to the nutritional quantity, considering the absence and lack of nutrients, and to the stock solution used in the preparation of the culture medium. Details on the concentration of each nutrient and effluent during the experiments are presented in Table 1.

The issue related to the nutritional medium involves its presence or absence, due to the hypothesis that the combination of microalgae and cyanobacteria can facilitate bioremediation. From this perspective, the breakdown of the molecules of the effluents in question could provide a nutritional medium for the microorganisms involved in the process.

The conditions presented in Table 1 for nutrient concentration, where:

• without addition, it presents 0%, that is, 0 mL of effluent;

• half presents 50%, that is, 50 mL of effluent;

• normal value 100%, that is, 100 mL of effluent;

• double 200%, that is, 200 mL of effluent.

These values are defined where the equivalence value is effluent:inoculum of 1:2, that is, the microorganisms used in this study can treat the effluents in question in these proportions for ideal conditions.

Table 1: Concentration of tested effluents

Source: Authors (2024).

Cultivation 1 (A), for example, was performed using 0% biodiesel (ethanol) wash water (0 mL), 20% inoculum quantity (200 mL) and without addition of nutritional medium (0 mL). Cultivation 1 (B) was performed using 0% biodiesel (ethanol) water (0 mL), 20% inoculum quantity (200 mL) and 50% (2.5 mL) of nutritional medium. Cultivation 2 (A) was performed using half (50 mL) biodiesel (ethanol) wash water, 20% inoculum quantity (200 mL) and without addition of nutritional medium (0 mL).

The other experiments used the same conditions presented above. Subsequently, cultivation was performed with 1% effluent (10 mL) and with 0.5% effluent (5 mL) to verify the biological conditions of the microalgae and cyanobacteria and their cell growth.

Every five days, cell growth determination analyses were performed using the spectrophotometric method, with absorbance measurements at a wavelength of 570 nm, until the 21st day of cultivation.

• • Results

The images obtained by the light microscope facilitated the initial identification of contamination in the culture, identification of microalgae and cyanobacteria at the genus level. The microscopic analyses were very important, since the culture samples are very small, requiring the adoption of tools for magnification and verification of cellular structures.

Co-culture involves the cultivation of several species together, mixing different types of microorganisms in the same location. To obtain images with greater resolution, temporary slides were made and observed under the Nikon Eclipse E200 Microscope. Through these, it was possible to verify the co-occurrence of the two distinct species in the co-culture (Chlorella sp. and Oscillatoria sp.), in addition to the formation of masses formed by the densification of microalgae cells with cyanobacterial filaments surrounded by a mucilaginous sheath, as shown in Figure 2.

Figure 2: Co-culture of microalgae and cyanobacteria at 400x magnification

Source: Authors (2024).

Co-culture has been gradually gaining ground in microbiology. Its use is an advantageous option when there is a harmonious interaction between the species that are actively participating in the co-culture. The microalgae-cyanobacteria consortium can perform mutualistic or competitive ecological interactions. Competitive interactions allow one species to benefit at the expense of another, reducing suitability, exploitation or parasitism. While cooperative interactions benefit from the activity of the other, with reciprocal interactions between microalgae that can promote the systemic absorption of nutrients and the growth of both in a harmonious way [8.].

To indicate the growth of microalgae and cyanobacteria in a co-culture with biodiesel wash water, the spectrophotometric method was used. Absorbance readings were performed up to the 20th day, or while the co-culture was alive. Monitoring was performed in the same way for all reactors, where the aliquot should represent as much of the real culture as possible.

Figure 3​​ shows the cell growth of the co-culture of test 4B, the characteristics of this culture were 200 mL of biodiesel water (ethanol), 2.5 mL of nutritional medium (Chu), 200 mL of inoculum and 597.5 mL of distilled water to have a total volume of 1 L.

Figure 3: Absorbance curve for cell growth of the microalgae Chlorella sp. in a co-cultivation system with a cyanobacteria species Oscillatoria sp. 21 days of cultivation (Trial 4B).

Source: Authors (2024).

The absorbance readings for days 0, 5, 10, 15 and 20 were 0.072; 0.097; 0.055; 0.032 and 0.017, respectively. The analyses demonstrate an exponential growth between days 0 and 5 and, subsequently, there was a decline in the absorbance readings, which in this case represents the cell death of the co-culture.

The low absorbance can be explained by the precariousness of nutrients in the biodiesel water, where the microalgae and cyanobacteria have no way to obtain the nutrients necessary for their basic biochemical needs.

Biodiesel wash water contains in its finais composition ethyl/methyl esters and oils (unconverted) in high concentrations, depending on the type of raw material used for its production, where these values can vary from 12% to 20% of the total volume and the pH of the effluent can vary between 9 and 10.5, which is harmful for co-cultures [9.].

The high levels of oil and biodiesel can make the culture medium highly toxic to microalgae and cyanobacteria, in which, in the absence of nutrients, they can consume their own carbohydrates and lipids due to the stress caused by the effluent. This factor alters their growth, their biochemical conditions and can lead to a stationary phase and later to the cell death of the entire culture [10.,11.].

Figure 4​​ shows the different types of cultivation in biodiesel wash water, with different amounts of effluent and nutritional medium, the answer is given in the number of days of cultivation to verify cell life and growth at the end of the cultivation.

It can be observed that in the cultures in biodiesel washing water with ethanol, methanol and ethanol + methanol in the proportions of 0% effluent and 50% nutritional medium, they were the only ones that presented 21 days of culture with satisfactory cell growth, however still below the absorbance readings for cyanobacteria and microalgae present in the literature.

Figure 4: Graphs showing the number of days of cultivation in different effluent proportions.

Source: Authors (2024).

 Because they are mixotrophic organisms, they perform photosynthesis and absorb nutrients present in the water. Therefore, all crops that had 50% of the nutritional medium present in the cultivation water had macro and micronutrients for their absorption, and this can be observed in a greater number of days of life and cell growth.

Crops that did not have nutritional medium had fewer days of cultivation, as there was cell death, where the microorganisms had no way to obtain nutrients other than from the organism itself, in this case, carbohydrates and lipids [12.].

Mixotrophic microorganisms require nutritional resources to satisfy their basic biochemical needs. There are ways to enhance these factors so that optimal conditions occur and by-products from their metabolism can be obtained [4.].

Biodiesel wash water does not contain nutrients that are satisfactory for microalgae in a form that they can absorb. The presence of oil that has not been transesterified, for example, which is present in this water, in addition to biodiesel residues, makes this effluent unviable for the cultivation of microalgae and cyanobacteria [9.,13.].

Other factors may also make this effluent unviable for the cultivation of microalgae and cyanobacteria, such as changes in pH due to the presence of the effluent and its toxicity. Many species of cyanobacteria are quite resistant to toxicity and adverse environments, but these microorganisms tend not to survive sudden changes in pH and high toxicity [14.].

Figure 5​​ shows that the co-cultivation that achieved greater adaptation with the effluent in smaller quantities and with the presence of ethanol, i.e., cell growth occurred, however, at a lower rate in the absence of effluent.

Figure 5​​ shows a 21-day cultivation with 20% inoculum with 50 mL of effluent (biodiesel wash water with ethanol) and 50% medium. The absorbance readings for days 0, 5, 10, 15 and 20 were respectively 0.069; 0.273; 0.301; 0.452 and 0.498. Some species of microalgae and cyanobacteria present readings similar to those presented in Figure ​​ 5, both for microalgae and cyanobacteria.

Figure 5: Absorbance curve for cell growth of the microalgae Chlorella sp. in a co-cultivation system with a cyanobacteria species Oscillatoria sp. 21 days of cultivation, with 50 mL of biodiesel wash water.

Source: Authors (2024).

 After all the cultivations were performed, it was observed that the biodiesel washing water remained in the upper part of the reactors in all cultivations, that is, the microalgae and cyanobacteria did not perform the biodegradation of the effluent satisfactorily, as shown in Figure 6. Another important factor, which could be observed in the cultivations with less than 21 days, was that cell death occurred in almost all reactors. The cells presented cellular rupture, loss of the functions of organelles that are very important in the biochemical relations of the cells, such as chloroplasts, and absence of coloration [15.].

Figure 6: Biodiesel wash water in the upper part of the reactor (A), then the dry reactor with the presence of biodiesel in the bottom of the reactor (B).

Source: Authors, 2024.

Another important consequence that relates cell death and the absence of biodegradation of the effluent in the reactors was the great contamination of fungi, shown in Figure ​​ 7. The reactors used in this study were of the tray type and closed with PVC film, in order to promote gas exchange, perforations were made in the PVC film, thus, when the cell death of microalgae and cyanobacteria occurred, the fungi had access to organic matter rich in lipids, carbohydrates and proteins, a humid and warm place, favoring contamination and its proliferation in most of the reactors.

Figure 7: Reactors A and B with contamination by fungi and insects.

Source: Authors (2024).

The ideal conditions for good development of fungi, that is, conditions favorable to life and reproducibility, are humidity around 90%, good nutritional quality and temperature between 23° and 30°C [16.].

Therefore, no new tests were carried out using the biodiesel wash water effluent, such as biomass and oil yield, production of biofuels and other co-products, nor even more precise analytical tests such as TGA and infrared, since the co-cultures did not present good results in terms of reproducibility, resistance and bioremediation of the chosen effluent.

• • Final considerations

It is concluded that cultivation in biodiesel wash water proved to be quite inefficient; after one week of cultivation, the microalgae and cyanobacteria did not survive the effluent contaminants. It is necessary to implement a new methodology and verify other proportions to obtain better results.

• • Acknowledgments

The authors express their gratitude for the financial support provided by the National Council for Scientific and Technological Development (CNPq), the Minas Gerais State Research Support Foundation (FAPEMIG) and the Coordination for the Improvement of Higher Education Personnel (CAPES), with the financing code 001.

• • Biographies

	Witter Duate Guerra PhD in Biofuel Science and Technology, also holding a master's degree in the same area from the Federal University of Uberlândia. Specialist in Genetics of Microorganisms and Biotechnology, in addition to having a degree in Biological Sciences from the Lutheran Institute of Higher Education of Itumbiara. http://lattes.cnpq.br/8951821384232273 https://orcid.org/0000-0002-9155-8047 .
	Jaqueline Elise Garcia Chiesa Master in Biofuel Science and Technology from the Federal University of Uberlândia. Graduated in Biological Sciences from the Lutheran Institute of Higher Education of Itumbiara. http://lattes.cnpq.br/9771109665383009 .
	Karolynne Gomes Albuquerque Graduated in Biological Sciences from the Lutheran Institute of Higher Education of Itumbiara. http://lattes.cnpq.br/9771109665383009 .
	Alexandre Matos Martins Agricultural Engineer, graduated from the State University of Montes Claros; PhD student in Biofuels - Federal University of Vales do Jequitinhonha and Mucuri, Master in Plant Production - Federal University of São João Del-Rey; Specialist in Environmental Management and Management from the Federal University of Lavras - UFLA. http://lattes.cnpq.br/7255938195800617 .
	Lucas Matheus da Rocha Graduated in Biological Sciences from the Federal University of Juiz de Fora, with a Master's degree in Biological Sciences from the Federal University of Rio de Janeiro and a PhD in Biological Sciences from the Postgraduate Program in Botany at the National Museum of the Federal University of Rio de Janeiro (2011) . He is currently Associate Professor at the Institute of Exact and Natural Sciences of Pontal-Campus Pontal of the Federal University of Uberlândia. http://lattes.cnpq.br/2684055663213328 .
	Anizio Marcio de Faria Bachelor's and licentiate degree in Chemistry from the Federal University of Viçosa in 2001, Master's in Agrochemistry from the Federal University of Viçosa in 2003, and Doctorate in Sciences, with a concentration in Chemistry, from the State University of Campinas in 2006. Completed post-doctoral research in Analytical Chemistry at the Institute of Chemistry of the State University of Campinas from 2007 to 2008. http://lattes.cnpq.br/5710906021234699 https://orcid.org/0000-0001-6915-8963 .
	Antonio Carlos Ferreira Batista Technician in Industrial Chemistry from UNAERP, Bachelor's degree in Chemistry from the University of São Paulo, Bachelor's degree in Chemistry with Technological Attributions from the University of São Paulo, Bachelor's degree in Chemistry from the University of São Paulo, Master's degree and PhD in Chemistry from the University from Sao Paulo. He is currently Associate Professor at the Institute of Exact and Natural Sciences of Pontal-Campus Pontal of the Federal University of Uberlândia. http://lattes.cnpq.br/6037406255124458 https://orcid.org/0000-0001-6313-4565 .