chemengineering

Article
A Phytochemical Approach to the Removal of Contaminants
from Industrial Dyeing Wastewater
Néstor A. Urbina-Suarez 1,2, * , Cristian J. Salcedo-Pabón 1 , Jefferson E. Contreras-Ropero 1 ,
German L. López-Barrera 1 , Janet B. García-Martínez 1 , Andrés F. Barajas-Solano 1 and
Fiderman Machuca-Martínez 2

1 Department of Environmental Sciences, Universidad Francisco de Paula Santander, Avenida Gran Colombia,
No. 12E-96, Cúcuta 540001, Colombia; [email protected] (C.J.S.-P.);
[email protected] (J.E.C.-R.); [email protected] (G.L.L.-B.);
[email protected] (J.B.G.-M.); [email protected] (A.F.B.-S.)
2 School of Natural Resources and Environment, Universidad del Valle, Cali 760015, Colombia;
[email protected]
* Correspondence: [email protected]

Abstract: This study investigates the influence of photoperiod and wastewater concentration on the
growth of microalgae and cyanobacteria for the removal of environmentally significant parameters
(COD, BOD, Cr, Fe, color, chlorides, nitrogen compounds, and phosphates) from dyeing wastewater.
A two-factor central composite design with surface response was employed, involving two algae
species (Chlorella and Scenedesmus sp.) and two cyanobacteria species (Hapalosiphon and Oscillatoria
sp.). The findings indicated that extended photoperiods (>13 h) and higher wastewater concentrations
(70–80% v/v) enhanced biomass production across all strains. However, Hapalosiphon and Chlorella
sp. (1.6 and 0.45 g/L) exhibited better tolerance to the wastewater’s high toxicity, resulting in higher
biomass concentrations and improved COD and BOD removal by Hapalosiphon sp. (75% and 80%,
Citation: Urbina-Suarez, N.A.; respectively). Further analysis of the obtained biomass revealed their potential applications. Among
Salcedo-Pabón, C.J.;
the cyanobacteria, Hapalosiphon sp. synthesized the highest concentrations of total proteins and lipids
Contreras-Ropero, J.E.;
(38% and 28% w/w, respectively), while Oscillatoria sp. displayed a high protein content (42% w/w).
López-Barrera, G.L.; García-Martínez,
In contrast, the algae demonstrated a strong propensity for storing substantial quantities of total
J.B.; Barajas-Solano, A.F.;
carbohydrates (65% and 57% w/w for Scenedesmus and Chlorella sp., respectively). These results
Machuca-Martínez, F. A
Phytochemical Approach to the
signify the feasibility of cultivating photosynthetic microorganisms in industrial dyeing wastewater
Removal of Contaminants from as a sustainable source of nutrients for targeted metabolite production.
Industrial Dyeing Wastewater.
ChemEngineering 2023, 7, 90. Keywords: dyeing wastewater; cyanobacteria; microalgae; biomass; metabolites; response surface
https://doi.org/10.3390/
chemengineering7050090

Academic Editor: Fausto Gallucci

1. Introduction
Received: 4 September 2023 Dyeing wastewater is an effluent produced during the dyeing, dismantling, washing,
Revised: 13 September 2023
and cleaning of garments in the textile industry. This industry uses large amounts of water
Accepted: 21 September 2023
and a wide range of dyes and synthetic chemicals at various stages of the production
Published: 28 September 2023
process [1]. This wastewater can contain multiple chemical substances, depending on
the products used. They are characterized by high levels of pollutants and the presence
of substances such as solvents, detergents, dyes, plasticizers, binders, volatile organic
Copyright: © 2023 by the authors.
compounds, surfactants, chlorobenzenes, phenols, pentachlorophenols, bleaching agents,
Licensee MDPI, Basel, Switzerland. and heavy metals [2,3] which may pose risks to both human well-being and the natural
This article is an open access article environment. It has been reported that the chemical compounds in dyeing effluents can be
distributed under the terms and toxic to aquatic life, negatively affecting marine ecosystems and reducing biodiversity. The
conditions of the Creative Commons effect on human well-being is substantial, resulting in liver and kidney impairment, skin
Attribution (CC BY) license (https:// inflammation, persistent bronchitis, nasal discomfort, the potential for cancer, and DNA
creativecommons.org/licenses/by/ harm. [4,5]. To minimize the impact of dyeing wastewater, it is essential to implement
4.0/). sound management practices, such as installing treatment systems. These systems can help

ChemEngineering 2023, 7, 90. https://doi.org/10.3390/chemengineering7050090 https://www.mdpi.com/journal/chemengineering

ChemEngineering 2023, 7, 90 2 of 18

eliminate or reduce wastewater pollutants before discharge, protecting the environment

and public health [6]. Various treatment processes have been developed to treat this
wastewater, including physical, chemical, and biological treatment processes to treat it
economically and efficiently [7]. The main physical treatment processes include filtration,
flocculation, and adsorption [8].
Methods based on filtration and flocculation are useful for effluents containing dis-
persed dyes. However, they are not very practical for wastewater containing reactive and
vat dyes, and the solids generated during treatment are considerable [9,10]. Methods like re-
verse osmosis (RO), nanofiltration (NF), and ultrafiltration (UF) have been employed for the
treatment of textile effluents, yielding favorable outcomes in the reduction in parameters
such as biological oxygen demand (BOD), chemical oxygen demand (COD), and color [11].
However, these processes have the disadvantage of high investment and operating costs,
the generation of other wastes, and the technological development necessary for their opera-
tion and maintenance, which are challenging to implement in less developed countries [12].
Adsorption processes have attracted much attention due to their efficiency in improving
the decolorization of wastewater. The adsorbents’ high affinity and recombination ability
are their most important properties. Some, such as activated carbon, have proven effective
for dyes like Acid Blue 25 (AB25) [13], but the difficulty of their regeneration and their price
limit their application [14].
Among chemical methods, oxidation processes are the most employed techniques for
breaking down dyes. Under environmental conditions, these processes can either partially
or entirely degrade the original toxicants and their chemical by-products, such as dyes,
pesticides, etc. Oxidation processes such as UV/H2 O2 have been shown to produce com-
plete decolorization after 20 min and a 63% removal of total organic carbon in 90 min [15];
similarly, combinations of TiO2 /UV/H2 O2 have achieved decolorizations of up to 64% with
illumination times of 100 min [16]. Other technologies, such as cavitation, can reduce color
content and improve the biodegradation index (BI) (BOD5 /COD ratio) [8]. According to
Mishra et al. [17], high removal rates of rhodamine can be achieved by coupling cavitation
with chemical reagents (H2 O2 , CCl4 , and Fenton reagent). The disadvantage of using these
methods is the generation of some by-products that cannot be recycled as waste or solids,
and finally, the development of technologies that sometimes limit their application.
On the other hand, biological processes usually use microorganisms that can thrive
under harsh conditions. The efficiency of elimination depends on the ratio between organic
load and dye, as well as on the concentration of microorganisms, temperature, oxygen
concentration in the system, and photoperiod [18]. The advantages of biological processes
include environmental compatibility, competitive cost, non-hazardous metabolites, and
reduced water consumption [19]. In recent times, microalgae and cyanobacteria have
emerged as sustainable remedies for eliminating harmful substances present in various
wastewater types, such as agricultural runoff [20], domestic wastewater [21], agroindustrial
wastewaters [22–25], and landfill leachate [26]. It was reported that Cosmarium species
could attenuate malachite green dye at temperatures of 5–45 ◦ C and pH 9, demonstrating
its ability to treat wastewater containing this pollutant [27].
Microalgae can remove total organic carbons, total nitrogen, and various dyes. Dhaouefi
et al. [28] demonstrated a reduction of 80% of orange 3 disperse dye and 75% of blue 1
disperse dye using an anoxic/aerobic photobioreactor at a contact time of 10 days [28].
There is limited work on the cultivation of microalgae and cyanobacteria in real dyeing
effluents, most being from synthetic effluents with a single dye; however, there are some
reports on the use of Chlorella sp. and Oscillatoria sp. strains for the treatment of dyeing
effluents. Nagaraj et al. [29] reported on the culture of S. pevalakii in textile wastewater at a
concentration of 60% (v/v) and found that the highest percentage of metabolites was found
in carbohydrates (50.1% w/w), followed by lipids (17.82% w/w), pigments (16.8% w/w),
and proteins (13.2% w/w). On the other hand, it was found for cyanobacteria that the
process of dye degradation involves several mechanisms that allow the decomposition and
elimination of various dyes of natural and synthetic origin. One of these processes is extra-
ChemEngineering 2023, 7, 90 3 of 18

cellular degradation, in which the dye molecules are degraded to smaller and less polluting
compounds with the help of extracellular enzymes produced by cyanobacteria [30]. It is
clear that both microalgae and cyanobacteria play an important role in different aquatic
ecosystems and can degrade various types of pollutants; however, effluent discharges with
dyes can inhibit their growth. Hence, although they can grow under these conditions,
it is possible that some dyes and compounds present in these effluents, given their low
biodegradability, may remain in the water body or, as a result of degradation, intermediate
compounds are generated that can affect aquatic life [31]. This aspect supports the need for
studies to evaluate the effect of factors such as wastewater concentration on the growth
and production of metabolites in microalgae and cyanobacteria in order to determine their
effectiveness in biotreating dye wastewater.
Currently, there are no reports on the effects of photoperiod and effluent concen-
tration using a response surface methodology on biomass production and recovery of
metabolites of interest in real dying wastewater; therefore, this work aims to contribute to
the knowledge of possible process optimization measures for variables such as photope-
riod and biomass production in nutrient reduction using microalgae and cyanobacteria in
recalcitrant effluents such as dying effluents.

2. Materials and Methods

2.1. Physicochemical Characterization of Dyeing Wastewater
The water sample was obtained from a textile and dyeing company in Cúcuta (Norte de
Santander, Colombia). Sampling was performed during the operating day, and a composite
sample was obtained. The sample was stored in 5 L amber bottles kept refrigerated at
4 ◦ C and taken to the laboratory at Universidad Francisco de Paula Santander (Cúcuta,
Colombia). The physicochemical characterization was done according to Standard Methods
23rd Edition [32]. The parameters analyzed are shown in Table 1.

Table 1. Physicochemical characterization of dyeing effluents.

Parameter Units Standard Methods Code

COD mg × L−1 5220C
BOD mg × L−1 5210B-4500-OG
Nitrates mg × L−1 4500-NO3 B
Nitrites mg × L−1 4500-NO2 B
Ammonia nitrogen mg × L−1 4500-NH3 F
Phosphates mg × L−1 4500-P C
Total Suspended Solids mg × L−1 2540D
Heavy Metals (Fe, Cr) mg × L−1 3111D
Sulfides mg × L−1 4500-S2 F
Chlorides mg × L−1 4500-ClB
Total Hardness mg × L−1 CaCO3 SM2340C
pH pH units 4500B
Conductivity µS × cm−1 2510B

2.2. Microorganisms
Two algae (Scenedesmus and Chlorella sp.) and two cyanobacteria (Hapalosiphon and
Oscillatoria sp.) were obtained from the INNOValgae collection (Universidad Francisco
de Paula Santander, Cúcuta, Colombia). Both algae were grown in liquid bold basal
medium, while the cyanobacteria were grown in liquid BG-11 medium. Every strain was
grown in 1 L GL-45 flask with 600 mL of culture media under a 12–12 h photoperiod,
110 µmol m−2 s−1 , mixed with filtered air enriched with 1% (v/v) of CO2 at a flow of
0.6 vvm and 25 ± 2 ◦ C [33].
ChemEngineering 2023, 7, 90 4 of 18

2.3. Experimental Design

A central composite design (CCD) with two levels (Table 2) was created on Design
Expert software (StatEase, Minneapolis, MN, USA, EEUU). The variables evaluated were
wastewater concentration and photoperiod; the response variable was biomass. The
experimental design resulted in 14 experiments conducted in triplicate. The dye water was
sedimented
ChemEngineering 2023, 7, x FOR PEER REVIEW for 30 min in a high-rate pilot settler in the UFPS unit operations laboratory
4 of 19
and then used for cultivation with the different strains. A 500 mL GL-45 flask with 300 mL
working volume (40 mL of algae plus 260 mL of wastewater) was used for each experiment.
Each
2.2. flask was grown for 20 days at 200 µmol m−2 s−1 , 25 ± 2 ◦ C and mixed with filtered
Microorganisms
air enriched with 1% (v/v) CO2 (Figure 1).
Two algae (Scenedesmus and Chlorella sp.) and two cyanobacteria (Hapalosiphon and
Oscillatoria sp.) weredesign
Table 2. Experimental obtained from the INNOValgae
for microalgae collection (Universidad Francisco de
and cyanobacteria.
Paula Santander, Cúcuta, Colombia). Both algae were grown in liquid bold basal medium,
while the cyanobacteria were grown
Experiment in liquid
Wastewater BG-11 medium. Every
Concentration strain was
Photoperiod grown in
Light/Dark
1 L GL-45 flask with 600 mL of culture media % v/v under a 12–12 h photoperiod,
Hours 110 µmol
m−2s−1, mixed 1with filtered air enriched with 201% (v/v) of CO2 at a flow of 0.6 vvm8 and 25 ±
2 °C [33] 2 50 8
3 20 21
2.3. Experimental
4 Design 50 21
A central5 composite design (CCD) with35two levels (Table 2) was created 14.5on Design
Expert software6 (StatEase, Minneapolis, MN,35USA, EEUU). The variables evaluated 14.5 were
7 35 14.5
wastewater concentration and photoperiod; the response variable was biomass. The ex-
8 56.21 14.5
perimental design
9
resulted in 14 experiments 35
conducted in triplicate. The dye14.5
water was
sedimented for 10 30 min in a high-rate pilot settler
35 in the UFPS unit operations
23.69 laboratory
and then used 11for cultivation with the different
35 strains. A 500 mL GL-45 flask14.5with 300 mL
working volume 12 (40 mL of algae plus 260 mL 35 of wastewater) was used for5.30 each experi-
ment. Each flask
13 was grown for 20 days at 35 200 µmol m−2s−1, 25 ± 2 °C and mixed with
14.5
14
filtered air enriched 13.78
with 1% (v/v) CO2 (Figure 1). 14.5

Figure 1. Diagram of the experimental process.

Figure 1. Diagram of the experimental process.
Table
2.4. 2. Experimental
Biomass designand
Concentration for Nutrient
microalgaeRemoval
and cyanobacteria.

Before inoculation,
Experiment the wastewater
Wastewater was autoclaved and
Concentration adjusted Light/Dark
Photoperiod to pH 7.0. For each
experiment, a 500 mL GL-45 flask%with v/v 250 mL of working volume was
Hoursused (25 mL of algae
plus 225 mL −2 −1
1 of wastewater). Each20flask was grown for 20 days under 8 200 µmol m s ,
◦
25 ± 2 C2and mixed using filtered50 air enriched with 1% (v/v) of CO2 8[33]. The wastewater
concentrations
3 and the light/dark cycles
20 were adjusted according to the21experimental design.
The ash-free
4 biomass was determined
50 according to Moheimani 21 Borowitzka [34].
and
After5 20 days, the biomass produced
35 was recovered through14.5
filtration in a 45 mm
GFC fiber6filter (5 mL per filter); each
35 flask was filtered 6 times. The filters were dried on
14.5
7 35 14.5
8 56.21 14.5
9 35 14.5
10 35 23.69
ChemEngineering 2023, 7, 90 5 of 18

a silica bed (60 ◦ C, 24 h). After drying, the filters were kept in the desiccator until they
reached a constant weight (±2 h), and then weighed [35]. After reaching constant weight,
the filters were incinerated in a muffle furnace (450 ◦ C, 5 h). Nitrates, phosphates, and COD
measurements were completed according to the standard methods for examining water
and wastewater [32]. For PO4 , the vanadomolybdophosphoric acid colorimetric method
was used (SM-4500-P C). For nitrates, the spectrophotometric UV-VIS detection method
was used (SM-4500-NO3 -B). For COD, the closed reflux colorimetric method was used
(SM-5220 D). The samples analyzed were taken in triplicate.
Total organic carbon (TOC) was determined using a TOC analyzer (Thermo Fisher
Scientific, Waltham, MA, USA). The operating conditions were a sample volume of 0.5 mL,
water chase volume of 1.0 mL, injection line rinse on, injection line rinse volume of 0.5 mL,
acid volume of 0.5 mL, ICS parge flow 200 mL min−1 , carrier gas delay time 0.40 min, ICS
parge time 50 min, detector sweep flow 500 mL min−1 , furnace sweep time 1.0 min, and
system flow 200 mL min−1 .

2.5. Carbohydrate Extraction and Quantification

A Falcon tube containing 0.5 mL of 1 M H2 SO4 and a filter with known biomass was
used for the experiment. The mixture was homogenized in a vortex for 2 min, and then
5 mL of 1 M H2 SO4 was added to it. The mixture was incubated in a water bath at 100 ◦ C
for 1 h. After that, the sample was centrifuged at 4000 rpm for 10 min at five ◦ C. In a
glass tube, 2 mL of the supernatant was collected, and 1 mL of 5% phenol was added. The
sample was shaken with a vortex at medium speed for 1 min and allowed to settle at room
temperature for 30 min before scanning at 485 nm [34]. A calibration curve ranging from 0
to 1.5 mg L1 was constructed to determine the concentration, and Equation (1) was used to
calculate total carbohydrates.

Total carbohydrates (mg × mL−1 ) = (0.0116 × OD485 ) + 0.0712 (1)

2.6. Total Lipids Extraction and Quantification

To determine the total lipid content of a sample, a filter with a known amount of
biomass was heated at 100 ◦ C for 10 min in a mixture of 100 mL ultrapure water and 2 mL
concentrated H2 SO4 . Then, freshly prepared sulfo-phospho-vanillin (SPV) was added to
the mixture and incubated at 37 ◦ C and 200 rpm for 15 min. Finally, the mixture was read at
a wavelength of 530 nm [35]. The entire process was repeated three times and a calibration
curve ranging from 0 to 1.5 mg L1 was generated. Equation (2) was used to calculate the
total lipid content of the sample.

Total lipids (µg) = (OD530 − 0.0236)/0.0106 (2)

2.7. Protein Extraction and Quantification

Total protein content was measured according to Slocombe et al. [36]. Three milliliters
of 24% trichloroacetic acid (TCA) wase added to a Falcon tube containing a filter with
known biomass. The mixture was heated at 95 ◦ C for 15 min in a water bath. After adding
9 mL of ultrapure water, the mixture was centrifuged (5000 rpm, 20 min, 4 ◦ C). The pellet
was then reconstituted in 0.5 mL of Lowry-D reagent and heated in a water bath (55 ◦ C,
60 min). Samples were centrifuged twice at (4500 rpm, 15 min). A total of 175 µL of the
supernatant was mixed with 3325 µL of Lowry D and incubated at room temperature for
10 min. Finally, 350 µL of Folin–Ciocalteu reagent was added and the sample stood at
room temperature for 30 min. Samples were read at 600 nm. Protein quantification was
performed according to Equation (3), and a calibration curve ranging from 0 to 5000 µg L−1
was constructed.

Total protein (mg L−1 ) = (2038.5 × OD600 ) + 59.706 (3)

ChemEngineering 2023, 7, 90 6 of 18

2.8. Total Carotenoids Extraction and Quantification

Falcon tubes containing filters with a fixed amount of biomass were utilized. To each
sample, 1 cm3 of 0.5 mm glass beads and 5 mL of ketone vehicle were added. The samples
were then homogenized for 3 min in a vortex at 100 rpm and centrifuged at 4500 rpm for
10 min at 4 ◦ C. The resultant supernatant was collected, and its absorbance was measured
at a wavelength of 450 nm [37]. By using Equation (4), the total concentration of carotenoids
was calculated.

Total carotenoids (mg/mL) = (OD450 × sample volume × 10)/2500 (4)

2.9. Phycobiliproteins Extraction and Quantification

To determine the phycocyanin concentration, filters containing known cyanobacterial
biomass were placed in Falcon tubes and suspended in 10 mL of a 0.15 M phosphate-buffered
solution with a pH of 7.0. Then, 2 g of glass beads were added, and the samples were allowed
to rest for 1 min before being shaken three times for 2 min each at maximum speed. The
samples were then stored at 4 ◦ C for 24 h, followed by centrifugation at 3400 rpm for 15 min.
Finally, the absorbance was measured at 620, 652, and 562 nm, and Equations (5)–(7) were
used to calculate the phycocyanin concentration as explained in [38].
C-PC (g L−1 ) = {[OD620 − (0.474 × OD652 )]}/5.34 (5)

A-PC (g L−1 ) = {[OD652 − (0.208 × OD620 )]}/5.09 (6)

PE (g L−1 ) = {[OD562 − (2.41 × C-PC) − (0.849 × APC)]}/9.62 (7)

3. Results and Discussions

3.1. Physicochemical Characterization of Dyeing Wastewater
Dyeing wastewater characterization is essential for ensuring compliance with envi-
ronmental regulations and protecting public health. In recent years, several studies have
been conducted to analyze the composition of dyeing wastewater and develop effective
treatment methods [36–38]. Table 3 shows the physicochemical characterization of the
dyeing effluents.

Table 3. Results of physicochemical characterization of wastewater.

Parameter Units This Research [38] [39] [40] [41] [42]

COD mg × L−1 974.7 ± 3.1 598 689 622 12,690 1700
BOD mg × L−1 290.33 ± 2.5 225.56 248 214.2 2667 782
Nitrates mg × L−1 42.95 ± 0.91 65 29.06 24.3 5.18 n/a
Nitrites mg × L−1 6.34 ± 0.09 1.25 n/a 3.23 n/a n/a
Ammonia nitrogen mg × L−1 18.45 ± 0.21 35.6 n/a 15.6 n/a 10.5
Phosphates mg × L−1 9.1 ± 0.72 n/a n/a 0.96 40.39 12.4
Total Suspended Solids mg × L−1 1345.31 ± 2.61 587.43 235 602.4 n/a n/a
Fe mg × L−1 1.98 ± 0.01 7.5 2.36 7.5 n/a 17.77
Cr mg × L−1 1.067 ± 0.01 n/a n/a 0.1 n/a 3.56
Chlorides mg × L−1 594 ± 0.12 850.67 2586 893.88 198.39 286
Total Hardness mg × L−1 CaCO3 612.21 ± 2.5 n/a n/a n/a n/a 1054
pH pH units 5.8 ± 0.1 6.1 7.5 5.98 4.94 6.25
Conductivity µS × cm−1 893.56 ± 3.4 1145 15.2 1302 1675 1180
n/a: not applicable.

The physicochemical characterization of the dyeing effluents shows low biodegradabil-

ity (0.29 BOD/DQO), a phenomenon similar to other reported studies [36,39,42]; similarly,
the concentrations of nitrogen compounds (nitrates, nitrites, and ammonia nitrogen) were
similar to the average ranges reported in other studies (Table 3). Phosphate concentra-
tions are within the range of other studies, where average phosphate concentrations of
ChemEngineering 2023, 7, 90 7 of 18

10–45.5 mg × L−1 were reported. Dyeing effluents can vary in their composition and con-
centration of contaminants, depending on the processes and chemicals used during the
dyeing process [39,43]. However, several studies have pointed to common physicochem-
ical characteristics of these effluents: in terms of pH, depending on the use of different
salts, bases, and acids, the pH can vary between acidic values (<5) and alkaline values
(>9) [36,44]; the COD concentration can range from 750 to 13,000 ppm, and most authors
cite BOD/CBD ratios of 0.15–0.4. These results are like those found in this work, indicating
a higher concentration of recalcitrant compounds that are not readily biodegradable by
microorganisms; therefore, the search for microorganisms that can degrade this type of
wastewater is a biotechnological potential not only for the treatment of these wastewaters,
but also for their use. The concentration of nitrogen compounds is related to the use of dyes
and nitrogen-containing chemicals in textile dyeing processes; nitrate concentrations have
been reported to be in the range of 35 to 80 ppm, and ammonium nitrogen concentrations
are usually between 25 and 65 ppm [45]. As for the concentration of phosphates, they are
typically present in dyeing wastewater at relatively low concentrations, generally ranging
from 0.5 to 20 mg × L−1 . The phosphates present in dyeing effluents are due to the use of
chemicals such as phosphate-based dyes, dyeing auxiliaries, detergents, and other additives
used during the process that contain these compounds.

3.2. Biomass Production and Nutrient Removal

The growth of both algae and cyanobacteria in textile wastewater can be influenced
by several factors that can favor or inhibit growth; much of this process depends on the
physicochemical properties of the wastewater, especially the availability of nutrients such
as NO3 − and PO4 −3 , which are necessary for their growth [46]. The presence of dyes in
these effluents can reduce the ability of algal strains to grow; compounds such as aromatic
amine, benzidine, and its derivatives can affect the growth rate, just as the concentration of
the dye affects the availability of light for microalgal species [47,48].
Cyanobacteria have been shown to grow at a higher rate than microalgae; these
organisms’ ability to fix nitrogen may enable their development, as physicochemical charac-
terization results showed low nitrate availability, explaining the low biomass concentration
of microalgae. There is limited work on bioremediation of actual dye wastewater by
microalgae and cyanobacteria. There are no reports on using Hapalosiphon sp. in these
processes. However, some authors have indicated that the process of degradation of these
dyes by cyanobacteria may involve absorption processes, extracellular binding by electro-
static interactions or van der Waals forces, and enzyme-mediated intracellular metabolism
in which the dye may be broken down into other, less toxic compounds, providing a
mechanism of detoxification of these compounds in terms of their cellular function [49,50].
In this study, the cyanobacteria showed more significant degradation and adjustment of
dyes in the residual water than the microalgae.
Table 4 presents the results of the ANOVA analysis for the four strains. In the case of
Chlorella sp., an F-value of 34.17 suggests that the model is significant, which implies that
such a large F-value cannot be due to the noise of the experiments. For Chlorella sp., wastew-
ater concentration and photoperiod are the most significant variables (p-value < 0.05). On
the other hand, the lack of fit F-value of 2.06 implies that the lack of fit is not significant
relative to the pure error. There is a 25.11% chance that such a large F-value for lack of fit is
due to noise.
For Scenedesmus sp. (Table 4), the photoperiod and residual water concentration with
the linear trend is significant, and the photoperiod with the quadratic trend is significant
in the model. Values greater than 0.1000 indicate that the model terms are not significant.
If there are many non-significant model terms (not counting those needed to support the
hierarchy), model reduction may improve the model. A lack of fit F-value of 0.04 implies
that the lack of fit is insignificant relative to the pure error. There is a 98.97% chance that
such a large F-value for lack of fit is due to noise.
ChemEngineering 2023, 7, 90 8 of 18

Table 4. ANOVA analysis for biomass of evaluated strains.

Strain Source Sum of Squares df Mean Square F-Value p-Value

Block 0.0008 1 0.0008
Model 0.0595 3 0.0198 34.17 <0.0001 *
A-Wastewater 0.0287 1 0.0287 49.57 <0.0001 *
B-Photoperiod 0.0210 1 0.0210 36.12 0.0002 *
Chlorella sp. A2 0.0098 1 0.0098 16.82 0.0027 *
Residual 0.0052 9 0.0006
Lack of Fit 0.0038 5 0.0008 2.06 0.2511 **
Pure Error 0.0015 4 0.0004
Cor Total 0.0655 13
Block 0.0063 1 0.0063
Model 0.2082 5 0.0416 83.78 <0.0001 *
A-Wastewater 0.0731 1 0.0731 147.11 <0.0001 *
B-Photoperiod 0.0624 1 0.0624 125.51 <0.0001 *
AB 0.0006 1 0.0006 1.26 0.2991 **
Scenedesmus sp. A2 0.0713 1 0.0713 143.50 <0.0001 *
B2 0.0000 1 0.0000 0.0913 0.7713 **
Residual 0.0035 7 0.0005
Lack of Fit 0.0001 3 0.0000 0.0355 0.9897 **
Pure Error 0.0034 4 0.0008
Cor Total 0.2179 13
Block 0.0206 1 0.0206
Model 0.9950 2 0.4975 85.89 <0.0001 *
A-Wastewater 0.0963 1 0.0963 16.62 0.0022 *
B-Photoperiod 0.8987 1 0.8987 155.15 <0.0001 *
Oscillatoria sp.
Residual 0.0579 10 0.0058
Lack of Fit 0.0264 6 0.0044 0.5581 0.7509 **
Pure Error 0.0315 4 0.0079
Cor Total 1.07 13
Block 0.0206 1 0.0206
Model 0.9950 2 0.4975 85.89 <0.0001 *
A-Wastewater 0.0963 1 0.0963 16.62 0.0022 *
B-Photoperiod 0.8987 1 0.8987 155.15 <0.0001 *
Hapalosiphon sp.
Residual 0.0579 10 0.0058
Lack of Fit 0.0264 6 0.0044 0.5581 0.7509 **
Pure Error 0.0315 4 0.0079
Cor Total 1.07 13
*—Significant. **—Not significant.

In the case of cyanobacteria, Oscillatoria sp. showed that the model’s F-value of 85.89 is
significant, indicating that there is only a 0.01% chance that this large F-value is due to noise.
If the p-value is less than 0.0500, the model terms are significant. For the case of Oscillatoria
sp., the residual water concentration and photoperiod are significant model terms. Values
greater than 0.1000 indicate that the terms used in the model are not statistically significant.
The lack of fit F-value of 0.56 suggests that the lack of fit is not significant compared to the
pure error. This means there is a 75.09% chance that such a high F-value for lack of fit is
due to random variation or noise.
Finally, the model for Hapalosiphon sp. was discovered to be significant (Table 4), with
an F-value of 206.30. This means there is only a 0.01% possibility that such a large F-value
is due to noise. When p-values are less than 0.0500, it indicates that the model terms are
significant. Both wastewater concentration and photoperiod, and the interaction between
these variables, were determined to be significant model terms. Values greater than 0.1000
indicate that the model terms are not significant. Similarly, the F-value for lack of fit of 5.73
implies that the lack of fit is not significant. There is only a 5.77% chance that such a large
F-value for lack of fit is due to noise.
 Finally, the model for Hapalosiphon sp. was discovered to be significant (Table 4), with
an F-value of 206.30. This means there is only a 0.01% possibility that such a large F-value
is due to noise. When p-values are less than 0.0500, it indicates that the model terms are
significant. Both wastewater concentration and photoperiod, and the interaction between
these variables, were determined to be significant model terms. Values greater than 0.1000
ChemEngineering 2023, 7, 90 indicate that the model terms are not significant. Similarly, the F-value for lack of fit9 ofof 18
5.73 implies that the lack of fit is not significant. There is only a 5.77% chance that such a
large F-value for lack of fit is due to noise.
Figure 2 shows the surface analysis for each of the microalgae and cyanobacteria
Figure 2 shows the surface analysis for each of the microalgae and cyanobacteria
strains in relation to the effects of photoperiod and effluent concentration on biomass pro-
strains in relation to the effects of photoperiod and effluent concentration on biomass
duction. It can be concluded that the best growth concentration is above the 50% mark
production. It can be concluded that the best growth concentration is above the 50% mark
(v/v) for all strains evaluated. The photoperiod also shows that the optimal growth ranges
(v/v) for all strains evaluated. The photoperiod also shows that the optimal growth ranges
could be 10 to 21 h of light/darkness. Based on the results obtained, a new experimental
could be 10
design wastocarried
21 h ofout
light/darkness. Based
using the results of theonoriginal
the results obtained,
experimental a newwhich
design, experimental
is pre-
design was carried
sented in Section 3.3.out using the results of the original experimental design, which is
presented in Section 3.3.

ChemEngineering 2023, 7, x FOR PEER REVIEW 10 of 19

(a) (b)

(c) (d)
Figure 2. Response surface analysis for biomass production of (a) Chlorella sp,. (b) Scenedesmus sp,.
Figure 2. Response surface analysis for biomass production of (a) Chlorella sp., (b) Scenedesmus sp.,
(c) Oscillatoria sp., and (d) Hapalosiphon sp.
(c) Oscillatoria sp., and (d) Hapalosiphon sp.
3.3.Validation
3.3. Validation of
of the
the Optimal
Optimal Conditions
Conditions
According to the response surface analysis results, the initial values were optimized
According to the response surface analysis results, the initial values were optimized
using a central composite design (CCD) with two levels of response surface analysis for
using a central composite design (CCD) with two levels of response surface analysis for
each of the strains studied. The values of the optimization design are shown in Table 5.
each of the strains studied. The values of the optimization design are shown in Table 5.
TableWith the optimization
5. Optimal experimental
initial conditions design
and conditions results,
for the ANOVAs
optimization were obtained for each
design.
of the strains evaluated and are presented in Table 6. The ANOVA results for Chlorella
the large F-value
sp. indicate significance;Wastewater is unlikely due to noise.Photoperiod
Additionally, p-values
Concentration
Strains
less than 0.05 indicate significant%model terms. In this case, Light/Dark
the linear behavior of the
v/v
wastewater and photoperiod and the quadratic behavior of the wastewater h are significant
model terms. Values above Low0.1000Medium
indicate High
that the modelLow terms Medium High With
are not significant.
regardChlorella sp.the F-value
to the fit, 50 for lack75of fit of 2.06
100 implies that
8 the lack12of fit is not16
significant
Scenedesmus
in relation to thesp. 50 There75is a 25.11%
pure error. 100chance that14 such a large
17 F-value20 for lack of
Oscillatoria sp. 50 75 100 17 20
fit is due to noise, indicating a positive and non-significant lack of fit. As for Scenedesmus 23
sp., an F-value ofsp.
Hapalosiphon 50
115.03 implies 75 the model
that 100 is significant;
17 there20 23 chance
is only a 0.01%

With the optimization experimental design results, ANOVAs were obtained for each
of the strains evaluated and are presented in Table 6. The ANOVA results for Chlorella sp.
indicate significance; the large F-value is unlikely due to noise. Additionally, p-values less
than 0.05 indicate significant model terms. In this case, the linear behavior of the
ChemEngineering 2023, 7, 90 10 of 18

that such a large F-value is due to noise. In this case, the linear and quadratic behavior of
the variables wastewater concentration and photoperiod are significant terms of the model.
The F-value for misfit of 5.04 implies a 7.62% chance of a misfit F-value due to noise.

Table 5. Optimal initial conditions and conditions for the optimization design.

Photoperiod
Wastewater Concentration
Light/Dark
Strains % v/v
h
Low Medium High Low Medium High
Chlorella sp. 50 75 100 8 12 16
Scenedesmus sp. 50 75 100 14 17 20
Oscillatoria sp. 50 75 100 17 20 23
Hapalosiphon sp. 50 75 100 17 20 23

Table 6. ANOVA analysis for biomass of the studied strains.

Strain Source Sum of Squares df Mean Square F-Value p-Value

Model 0.0595 3 0.0198 34.17 <0.0001 *
A-Wastewater 0.0287 1 0.0287 49.57 <0.0001 *
Chlorella sp. B-Photoperiod 0.0210 1 0.0210 36.12 0.0002 *
A2 0.0098 1 0.0098 16.82 0.0027 *
Lack of Fit 0.0038 5 0.0008 2.06 0.2511 **
Model 0.2082 5 0.0416 83.78 <0.0001 *
A-Wastewater 0.0731 1 0.0731 147.11 <0.0001 *
B-Photoperiod 0.0624 1 0.0624 125.51 <0.0001 *
Scenedesmus sp. AB 0.0006 1 0.0006 1.26 0.2991 *
A2 0.0713 1 0.0713 143.50 <0.0001 *
B2 0.0000 1 0.0000 0.0913 0.7713 **
Lack of Fit 0.0001 3 0.0000 0.0355 0.9897 **
Model 1.31 5 0.2621 133.19 <0.0001 *
A-Wastewater 0.1375 1 0.1375 69.87 <0.0001 *
B-Photoperiod 0.0398 1 0.0398 20.23 0.0028 *
Oscillatoria sp. AB 0.0047 1 0.0047 2.38 0.1665 **
A2 0.7865 1 0.7865 399.60 <0.0001 *
B2 0.4839 1 0.4839 245.86 <0.0001 *
Lack of Fit 0.0081 3 0.0027 1.91 0.2687 **
Model 1.49 5 0.2977 87.50 <0.0001 *
A-Wastewater 0.1540 1 0.1540 45.25 0.0003 *
B-Photoperiod 0.0484 1 0.0484 14.24 0.0070 *
Hapalosiphon sp.
A2 0.0020 1 0.0020 0.5951 0.4657 *
B2 0.9770 1 0.9770 287.10 <0.0001 *
Lack of Fit 0.0257 5 0.0051 5.73 0.0577 **
*—Significant. **—Not significant.

In Oscillatoria sp., it is evident that the model F-value of 133.19 implies that the model
is significant; there is only a 0.01% chance that such a large F-value is due to noise. In this
case, the linear and quadratic behavior of the wastewater concentration and photoperiod
are significant terms of the model. In this case, the F-value for lack of fit of 1.91 implies
that the lack of fit is not significant in relation to the pure error. There is a 26.87% chance
that such a large F-value of misfit is due to noise. Finally, for Hapalosiphon sp., the model
F-value of 115.03 indicated that the model is significant; there is only a 0.01% chance that
such a large F-value is due to noise. It was found that the linear and quadratic behavior of
wastewater concentration and photoperiod are significant terms of the model. The lack of
fit F-value of 5.04 implies a 7.62% chance of such a significant lack of fit F-value being due
to noise.
ChemEngineering 2023, 7, 90 11 of 18

ChemEngineering 2023, 7, x FOR PEER REVIEW 12 of 19

Figure 3 shows the results of the response surface analysis for the optimization of the
data obtained in the original experimental design.

(a) (b)

(c) (d)
Figure 3. Optimization of wastewater concentration and photoperiod via response surface analysis
Figure 3. Optimization of wastewater concentration and photoperiod via response surface analysis for
for biomass production for (a) Chlorella sp., (b) Scenedesmus sp., (c) Oscillatoria sp., and (d) Hapalosi‐
phon sp.production for (a) Chlorella sp., (b) Scenedesmus sp., (c) Oscillatoria sp., and (d) Hapalosiphon sp.
biomass

The
Theresponse
responsesurface
surface allowed ustotoobtain
allowed us obtainthe
thevariables
variablesto to confirm
confirm thethe process
process condi-
condi-
tions (Table 7).
tions (Table 7).

Table7.7. Optimal
Table conditionswere
Optimal conditions were obtained
obtained from
from response
response surface
surface analysis
analysis using Expert
using Design Designsoft-
Expert
software.
ware.

Photoperiod
Photoperiod
Wastewater Concentration
Wastewater Concentration
Strain
Strain Light/Dark
Light/Dark
% v/v
% v/v
hh
Chlorella
Chlorellasp.
sp. 80
80 13.5
13.5
Scenedesmus
Scenedesmussp. sp. 81
81 18.5
18.5
Oscillatoria sp.
Oscillatoria sp. 75
75 19.2
19.2
Hapalosiphonsp.
Hapalosiphon sp. 75
75 19
19

Tenreplicates
Ten replicates were
were performed
performed to toconfirm
confirmthetheconditions
conditionsdescribed in Table
described 7, and
in Table a a
7, and
T-Test Student ANOVA statistical analysis was performed using Prism-GraphPad
T-Test Student ANOVA statistical analysis was performed using Prism-GraphPad software. soft-
ware. 4Figure
Figure shows4 shows the results
the results of the of the verification
verification of theofoptimal
the optimal conditions.
conditions.
 ChemEngineering 2023, 7, x FOR PEER REVIEW 13 of 19
ChemEngineering 2023, 7, 90 12 of 18

Figure
Figure4.4.Validation
Validationofofoptimal
optimalconditions
conditions forfor
biomass production
biomass onon
production Chlorella sp.sp.
Chlorella (a),(a),
Scenedesmus
Scenedesmus sp.
sp.
(b),(b), Hapalosiphon
Hapalosiphon sp.sp.(c),
(c),and
andOscillatoria
Oscillatoriasp.
sp. (d).
(d).

Biomass
Biomassproduction
production is is
generally
generallyrelated
relatedto the dyedye
to the degradation
degradationrate; rate;
the type
the and
type and
concentration of dye significantly affects biomass production. However,
concentration of dye significantly affects biomass production. However, textile effluents textile effluents
have
havedifferent
differenttypes
typesandandconcentrations
concentrations of dyes thatthat
of dyes affect the biomass
affect of microalgae
the biomass and and
of microalgae
cyanobacteria [51]. Although there are few studies on real dye effluents,
cyanobacteria [51]. Although there are few studies on real dye effluents, reports on reports on the use
the use
of microalgae and cyanobacteria in textile effluents have shown that
of microalgae and cyanobacteria in textile effluents have shown that the concentration the concentration of of
dye
dyeininthe
thesystem
systemimproves
improvesthe theremoval
removalrate ratedueduetotothe
theincreased
increased contact
contactopportunities
opportunities be-
between
tween the the
dyedye moleculesand
molecules andthethe microalgae
microalgae and
and cyanobacteriacells
cyanobacteria cellsduring
duringthethe bio-
biosorption
sorption process and is also a driving force for overcoming all the barriers for mass trans-
process and is also a driving force for overcoming all the barriers for mass transfer of the
fer of the dye molecules between the cells [28,50]. Degradation of dyes requires a source
dye molecules between the cells [28,50]. Degradation of dyes requires a source of nitrogen
of nitrogen and phosphorus and, depending on the type of metabolism (autotrophy, mix-
and phosphorus and, depending on the type of metabolism (autotrophy, mixotrophy, and
otrophy, and heterotrophy), a source of organic carbon [40,52]. In this work, it was shown
heterotrophy), a source of organic carbon [40,52]. In this work, it was shown that nitrate
that nitrate concentration in wastewater affects the growth of Scenedesmus and Chlorella
concentration in wastewater affects the growth of Scenedesmus and Chlorella sp., while it has
sp., while it has no effect in the case of cyanobacteria since they can fix nitrogen. Finally,
itnohas
effect
beeninpointed
the caseoutof cyanobacteria
that the effluent since they can fix
concentration andnitrogen. Finally,
light cycle affectit the
hasbiomass
been pointed
out that theprocess
production effluent ofconcentration
both microalgae and andlight cycle affect[53];
cyanobacteria the biomass
this workproduction
has shown process
that of
both microalgae and cyanobacteria [53]; this work has shown that
the optimization of these parameters increased the biomass production in all the strains the optimization of these
parameters
evaluated. increased
Figure 5 shows thethe
biomass
removalproduction
of contaminants in allfrom
the strains evaluated.
the conditions Figure
by each 5 shows
strain.
the removal of contaminants from the conditions by each strain.
As shown in Figure 5, the removals of most parameters were above 50%, and the
strain with the best pollutant removal was Hapalosiphon sp. (Figure 5) which effectively
removed COD (75%), nitrates (97%), ammonium (88%), TDS (90%), Fe (91.4), and Cr
(92.3%). COD, nitrogen, and phosphorous are critical indicators for evaluating the quality
of dyeing wastewater; the assimilation of nitrogen and phosphorous compounds by mi-
croalgae cyanobacteria helps the metabolism and growth of these organisms and, therefore,
consumes any organic substrate and reduces the COD present in the wastewater [54]. This
work demonstrated 85% higher removals of nitrogen and phosphorus compounds, leading
to COD removals above 65% for the evaluated strains.
ChemEngineering 7, 907, x FOR PEER REVIEW
2023,2023,
ChemEngineering 14 of1319of 18

Figure
5. 5. Contaminantremoval
Contaminant removalpercentage
percentage of
of COD
COD (a),
(a), BOD
BOD (b),
(b),NO
NO33(c),
(c),NO
NO2 (d), NH4 (e), PO4 (f),
Figure 2 (d), NH4 (e), PO4 (f),
TSS (g), Fe (h), Cr (i), and Cl (j). Total hardness (k) and conductivity (l).
TSS (g), Fe (h), Cr (i), and Cl (j). Total hardness (k) and conductivity (l).
As shown
3.4. Production in Figure 5,ofthe
of Metabolites removals of most parameters were above 50%, and the
Interest
strain with the best pollutant removal was Hapalosiphon sp. (Figure 5) which effectively
Figure 6 shows the percentage of metabolites produced by Scenedesmus, Chlorella,
removed COD (75%), nitrates (97%), ammonium (88%), TDS (90%), Fe (91.4), and Cr
Oscillatoria, and Hapalosiphon sp. grown in industrial dyeing wastewater according to the
(92.3%). COD, nitrogen, and phosphorous are critical indicators for evaluating the quality
biomass production
of dyeing wastewater; optimization conditions
the assimilation obtained.
of nitrogen Evidently, the
and phosphorous highest production
compounds by mi-
was obtained in the strains of cyanobacteria: 38.5% for Hapalosiphon
croalgae cyanobacteria helps the metabolism and growth of these organisms sp. and
and,42.3%
there- for
Oscillatoria sp.; inany
fore, consumes carbohydrates, the and
organic substrate microalgae strains
reduces the CODobtained
present inthe
thehighest production
wastewater [54].
percentages: 65. 38% for Scenedesmus sp. and 57.68% for Chlorella sp. For lipids, the strain
Hapalosiphon sp. showed the highest accumulation (25.88%), and finally, the cyanobacteria
showed the highest pigment concentration. The variation in metabolite production rates
between microalgae and cyanobacteria primarily arises from the distinct metabolic traits
 cillatoria, and Hapalosiphon sp. grown in industrial dyeing wastewater according to the bi-
omass production optimization conditions obtained. Evidently, the highest production
was obtained in the strains of cyanobacteria: 38.5% for Hapalosiphon sp. and 42.3% for Os‐
cillatoria sp.; in carbohydrates, the microalgae strains obtained the highest production per-
centages: 65. 38% for Scenedesmus sp. and 57.68% for Chlorella sp. For lipids, the strain
ChemEngineering 2023, 7, 90 Hapalosiphon sp. showed the highest accumulation (25.88%), and finally, the cyanobacteria 14 of 18
showed the highest pigment concentration. The variation in metabolite production rates
between microalgae and cyanobacteria primarily arises from the distinct metabolic traits
exhibited
exhibited by by these
these organisms
organisms[55].
[55].Factors
Factorssuch as as
such biomass density,
biomass molecular
density, weight,
molecular the the
weight,
type of algal biomass, species variations, metabolic activity, growth stage, environmental
type of algal biomass, species variations, metabolic activity, growth stage, environmental
circumstances, and the presence of nutrients in the wastewater can influence the levels
circumstances, and the presence of nutrients in the wastewater can influence the levels and
and buildup of metabolites [56]. There are few studies on the cultivation of microalgae
buildup of metabolites [56]. There are few studies on the cultivation of microalgae and
and cyanobacteria in real dyeing effluents, while most studies are on synthetic effluents
cyanobacteria in real dyeing effluents, while most studies are on synthetic effluents using a
using a single dye; however, there are some reports on the use of Chlorella sp. and Oscilla‐
single dye; however, there are some reports on the use of Chlorella sp. and Oscillatoria sp.
toria sp. strains for the treatment of dyeing effluents.
strains for the treatment of dyeing effluents.

Figure 6.
Figure 6. Metabolite
Metabolite production
production of
of Chlorella
Chlorella sp.,
sp., Scenedesmus
Scenedesmussp.,
sp.,Oscillatoria
Oscillatoriasp.,
sp.,and
andHapalosiphon
Hapalosiphon sp.
sp.
Nagaraj et al. [29] reported on the cultivation of S. pevalakii in textile wastewater at
Nagaraj et al.
a concentration of[29]
60%reported
and found on the
thatcultivation
the highestof S.percentage
pevalakii in of
textile wastewater
metabolites was atfound
a
concentration of 60% and found that the highest percentage of metabolites
in carbohydrates (50.1% w/w), followed by lipids (17.82% w/w), pigments (16.8% w/w), was found in
carbohydrates
and proteins (13).(50.1% w/w),
In this followed
work, by lipids
proteins were(17.82%
found to w/w), pigments
be the (16.8%
metabolite w/w),
with theand
highest
proteins (13). In this work, proteins were found to be the metabolite with
production for Oscillatoria sp. (42.4% w/w), while for the other metabolites, the results the highest pro-were
ductionexcept
similar, for Oscillatoria sp. (42.4%
for pigments, where w/w),
thewhile for the other
concentration metabolites,
of Oscillatoria wasthelower.
resultsInwere
the case
similar, except for pigments, where the concentration of Oscillatoria
of Chlorella, Tamil et al. [57] reported the highest biomass production of C. was lower. In vulgaris
the case in a
of Chlorella, Tamil et al. [57] reported the highest biomass production of C. vulgaris in a
50% dilution of textile effluent, achieving carbohydrate production percentages of 34.6%
50% dilution of textile effluent, achieving carbohydrate production percentages of 34.6%
w/w and lipid percentages of 13.88% w/w. Compared with the results obtained in this
w/w and lipid percentages of 13.88% w/w. Compared with the results obtained in this
study, it was found that the accumulation of carbohydrates (54.7% w/w) was higher, and
study, it was found that the accumulation of carbohydrates (54.7% w/w) was higher, and
the concentration of lipids was similar. Regarding Scenedesmus, the existing reports refer to
the concentration of lipids was similar. Regarding Scenedesmus, the existing reports refer
biomass production; however, Selvan et al. [58] reported on the growth of Scenedesmus sp.
to biomass production; however, Selvan et al. [58] reported on the growth of Scenedesmus
and Desmosdesmus
sp. and Desmosdesmus sp. strains in synthetic
sp. strains water
in synthetic withwith
water dyes, where
dyes, wherethe the
percentage
percentageof protein
of
achieved w/w Scenedesmus sp. and 1.5% w/w;
protein achieved was 1.8% w/w for Scenedesmus sp. and 1.5% w/w; in this study, the per- of
was 1.8% for in this study, the percentage
protein
centageproduction was higher than
of protein production that (10.05%
was higher w/w);
than that Arutselvan
(10.05% et al. [59] reported
w/w); Arutselvan a lipid
et al. [59]
accumulation of 39% w/w for S. dimorphus strain, a higher value than that
reported a lipid accumulation of 39% w/w for S. dimorphus strain, a higher value than that achieved in this
study for the Scenedesmus sp. strain (15.63% w/w). Regarding Hapalosiphon sp., there are still
no reports in the literature about its use in the treatment of wastewater from the dyeing and
textile industries. It is worth highlighting the enrichment of proteins (39.3% w/w) and lipids
(25.88% w/w), which reached the highest concentration in this study. Some studies have
reported that the production of biomass and metabolites in microalgae and cyanobacteria
largely depends on the degradation capacity of the various dyes that make up the dyeing
effluent and become sources of nitrogen, carbon, and phosphorus [56]. In genera such as
Chlorella sp. and Scenedesmus sp., it has been found that degradation of dyes can occur
in two ways: a phase I involving hydrolysis reactions, oxidation, or reduction processes
involving enzymes such as hydroxylases, carboxylases, monooxygenases, dioxygenases,
and decarboxylases, And a phase II including biodegradation reactions by enzymes such
as hydrolases, catalase transferases, glutamyl-tRNA dehydrogenase, malate/pyruvate
mono(di)-oxygenase, dehydratases, catalases, reductases, glutathione S-transferases, and
pyrophosphate carboxylase/decarboxylase [58,60]. With respect to cyanobacteria, it was
found that the process of dye degradation involves several mechanisms that allow the
degradation and elimination of various dyes of natural and synthetic origin. One of the
ChemEngineering 2023, 7, 90 15 of 18

processes is extracellular degradation, in which the dye molecules are degraded to smaller
and less contaminating compounds with the help of extracellular enzymes produced by
cyanobacteria. Another process is intracellular degradation, in which enzymes of the
genus azoreductases attack the azo bonds present in the dyes and decompose the dye
molecules [30,51].

4. Conclusions
Recently, microalgae and cyanobacteria have been proposed as sustainable solutions
for removing pollutant compounds in wastewater. Studies have also been initiated on
using these microorganisms in dyeing wastewater due to their ability to tolerate extreme
environments, assimilate compounds in sewage, and utilize the biomass produced due
to the metabolites produced: lipids, proteins, phytohormones, carbohydrates, and pig-
ments. In this study, the use of microalgal strains (Scenedesmus sp. and Chlorella sp.) and
cyanobacteria (Oscillatoria sp. and Hapalosiphon sp.) for bioremediation of dyeing wastewa-
ter, biomass production, and metabolites of interest were tested, analyzing the effects of
the photoperiod and wastewater concentration. The results showed that for most of the
strains studied, longer light cycles of more than 18 h favored growth, except for Chlorella
sp., whose photoperiod was 13 h/11 h light/dark. A residual water concentration of
75% v/v proved optimal for the studied strains’ growth. The strain exhibiting the best
pollutant removal was Hapalosiphon sp., which effectively removed COD (75%), nitrates
(97%), ammonium (88%), TDS (90%), Fe (91.4), and Cr (92.3%). In terms of metabolite
production, the highest production of proteins was obtained in the cyanobacterial strains,
38.5% for Hapalosiphon sp. and 42.3% for Oscillatoria sp.; in the case of carbohydrates, the
microalgae strains obtained the highest production percentage, 65.38% for Scenedesmus sp.
and 57.68% for Chlorella sp. Regarding lipids, the strain Hapalosiphon sp. showed the highest
accumulation (25.88%), and finally, in terms of pigment concentration, the cyanobacteria
showed the highest accumulation. The cyanobacteria have greater adaptability due to their
ability to fix nitrogen. Their potential use depends on the metabolite to be recycled, so this
work provides a basis for further research on the utilization of this wastewater through
optimization processes to obtain a metabolite of interest. It is, therefore, necessary to
continue researching the use of these microorganisms as a biotechnological strategy for the
treatment of dyeing effluents; it is essential to evaluate the treated effluent by investigating
the compounds that may remain as residual or intermediate products generated during
the treatment bioprocess, to determine not only the use of the biomass generated, but
also the possibility of reincorporating the treated effluents into the production process,
guaranteeing the sustainability of the process.

Author Contributions: Conceptualization, F.M.-M.; Data curation, A.F.B.-S., J.B.G.-M., N.A.U.-S. and
G.L.L.-B.; Funding acquisition, F.M.-M.; Investigation, N.A.U.-S. and C.J.S.-P.; Resources, A.F.B.-S.
and F.M.-M.; Software, J.E.C.-R., N.A.U.-S., J.B.G.-M. and A.F.B.-S.; Supervision, A.F.B.-S. and F.M.-M.;
Writing—original draft, N.A.U.-S. and C.J.S.-P.; Writing review and editing, A.F.B.-S., G.L.L.-B. and
F.M.-M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by Universidad del Valle (Colombia) with the project “Evalu-
ation of a coupled system for advanced oxidation of industrial effluents and production of microal-
gae to obtain high value-added metabolites. ID CI 21144” and Newton Fund Institutional Links,
ID 527624805.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to express our sincere gratitude to Universidad del Valle and
Universidad Francisco de Paula Santander for the equipment for this research.
Conflicts of Interest: The authors declare no conflict of interest.
ChemEngineering 2023, 7, 90 16 of 18

References
1. Ceretta, M.B.; Vieira, Y.; Wolski, E.A.; Foletto, E.L.; Silvestri, S. Biological Degradation Coupled to Photocatalysis by
ZnO/Polypyrrole Composite for the Treatment of Real Textile Wastewater. J. Water Process Eng. 2020, 35, 101230. [CrossRef]
2. Hubadillah, S.K.; Othman, M.H.D.; Tai, Z.S.; Jamalludin, M.R.; Yusuf, N.K.; Ahmad, A.; Rahman, M.A.; Jaafar, J.; Kadir,
S.H.S.A.; Harun, Z. Novel Hydroxyapatite-Based Bio-Ceramic Hollow Fiber Membrane Derived from Waste Cow Bone for Textile
Wastewater Treatment. Chem. Eng. J. 2020, 379, 122396. [CrossRef]
3. Yuan, Y.; Ning, X.A.; Zhang, Y.; Lai, X.; Li, D.; He, Z.; Chen, X. Chlorobenzene Levels, Component Distribution, and Ambient
Severity in Wastewater from Five Textile Dyeing Wastewater Treatment Plants. Ecotoxicol. Environ. Saf. 2020, 193, 110257.
[CrossRef] [PubMed]
4. Chhikara, S.; Hooda, A.; Rana, L.; Dhankhar, R. Chromium (VI) Biosorption by Immobilized Aspergillus Niger in Continuous
Flow System with Special Reference to FTIR Analysis. J. Environ. Biol. 2010, 31, 561–566. [PubMed]
5. Daneshvar, E.; Zarrinmehr, M.J.; Kousha, M.; Hashtjin, A.M.; Saratale, G.D.; Maiti, A.; Vithanage, M.; Bhatnagar, A. Hexavalent
Chromium Removal from Water by Microalgal-Based Materials: Adsorption, Desorption and Recovery Studies. Bioresour. Technol.
2019, 293, 122064. [CrossRef] [PubMed]
6. Chung, K.T. Mutagenicity and Carcinogenicity of Aromatic Amines Metabolically Produced from Azo Dyes. J. Environ. Sci.
Health Part C 2008, 18, 51–74. [CrossRef]
7. Kumar, P.; Sumangala, B. Decolorization of Azo Dye Red 3BN by Bacteria. Int. Res. J. Biol. Sci. 2012, 1, 46–52.
8. Holkar, C.R.; Jadhav, A.J.; Pinjari, D.V.; Mahamuni, N.M.; Pandit, A.B. A Critical Review on Textile Wastewater Treatments:
Possible Approaches. J. Environ. Manag. 2016, 182, 351–366. [CrossRef]
9. Liang, C.Z.; Sun, S.P.; Li, F.Y.; Ong, Y.K.; Chung, T.S. Treatment of Highly Concentrated Wastewater Containing Multiple Synthetic
Dyes by a Combined Process of Coagulation/Flocculation and Nanofiltration. J. Membr. Sci. 2014, 469, 306–315. [CrossRef]
10. Yeap, K.L.; Teng, T.T.; Poh, B.T.; Morad, N.; Lee, K.E. Preparation and Characterization of Coagulation/Flocculation Behavior of a
Novel Inorganic–Organic Hybrid Polymer for Reactive and Disperse Dyes Removal. Chem. Eng. J. 2014, 243, 305–314. [CrossRef]
11. Chollom, M.N.; Rathilal, S.; Pillay, V.L.; Alfa, D. The Applicability of Nanofiltration for the Treatment and Reuse of Textile
Reactive Dye Effluent. Water SA 2015, 41, 398–405. [CrossRef]
12. Koyuncu, I.; Güney, K. Membrane-Based Treatment of Textile Industry Wastewaters. Encycl. Membr. Sci. Technol. 2013, 1–12.
[CrossRef]
13. Auta, M.; Hameed, B.H. Preparation of Waste Tea Activated Carbon Using Potassium Acetate as an Activating Agent for
Adsorption of Acid Blue 25 Dye. Chem. Eng. J. 2011, 171, 502–509. [CrossRef]
14. Galán, J.; Rodríguez, A.; Gómez, J.M.; Allen, S.J.; Walker, G.M. Reactive Dye Adsorption onto a Novel Mesoporous Carbon. Chem.
Eng. J. 2013, 219, 62–68. [CrossRef]
15. Zuorro, A.; Lavecchia, R. Evaluation of UV/H2 O2 Advanced Oxidation Process (AOP) for the Degradation of Diazo Dye Reactive
Green 19 in Aqueous Solution. New Pub Balaban 2014, 52, 1571–1577. [CrossRef]
16. Gupta, V.K.; Jain, R.; Mittal, A.; Saleh, T.A.; Nayak, A.; Agarwal, S.; Sikarwar, S. Photo-Catalytic Degradation of Toxic Dye
Amaranth on TiO(2)/UV in Aqueous Suspensions. Mater. Sci. Eng. C Mater. Biol. Appl. 2012, 32, 12–17. [CrossRef] [PubMed]
17. Mishra, K.P.; Gogate, P.R. Intensification of Degradation of Rhodamine B Using Hydrodynamic Cavitation in the Presence of
Additives. Sep. Purif. Technol. 2010, 75, 385–391. [CrossRef]
18. Wang, Z.; Xue, M.; Huang, K.; Liu, Z.; Wang, Z.; Xue, M.; Huang, K.; Liu, Z. Textile Dyeing Wastewater Treatment. Adv. Treat.
Text. Effl. 2011, 5, 91–116. [CrossRef]
19. Hayat, H.; Mahmood, Q.; Pervez, A.; Bhatti, Z.A.; Baig, S.A. Comparative Decolorization of Dyes in Textile Wastewater Using
Biological and Chemical Treatment. Sep. Purif. Technol. 2015, 154, 149–153. [CrossRef]
20. Castellanos-Estupiñan, M.A.; Carrillo-Botello, A.M.; Rozo-Granados, L.S.; Becerra-Moreno, D.; García-Martínez, J.B.; Urbina-
Suarez, N.A.; López-Barrera, G.L.; Barajas-Solano, A.F.; Bryan, S.J.; Zuorro, A. Removal of Nutrients and Pesticides from
Agricultural Runoff Using Microalgae and Cyanobacteria. Water 2022, 14, 558. [CrossRef]
21. Zuorro, A.; García-Martínez, J.B.; Barajas-Solano, A.F. The Application of Catalytic Processes on the Production of Algae-Based
Biofuels: A Review. Catalysts 2021, 11, 22. [CrossRef]
22. Guiza-Franco, L.; Orozco-Rojas, L.G.; Sanchez-Galvis, M.; Garcia-Martinez, J.B.; Barajas-Ferreira, C.; Zuorro, A.; Barajas-Solano,
A.F. Production of Chlorella Vulgaris Biomass on UV-Treated Wastewater as an Alternative for Environmental Sustainability on
High-Mountain Fisheries. Chem. Eng. Trans. 2018, 64, 517–522. [CrossRef]
23. Quintero-Dallos, V.; García-Martínez, J.B.; Contreras-Ropero, J.E.; Barajas-Solano, A.F.; Barajas-Ferrerira, C.; Lavecchia, R.; Zuorro,
A. Vinasse as a Sustainable Medium for the Production of Chlorella Vulgaris UTEX 1803. Water 2019, 11, 1526. [CrossRef]
24. García-Martínez, J.B.; Sanchez-Tobos, L.P.; Carvajal-Albarracín, N.A.; Barajas-Solano, A.F.; Barajas-Ferreira, C.; Kafarov, V.;
Zuorro, A. The Circular Economy Approach to Improving CNP Ratio in Inland Fishery Wastewater for Increasing Algal Biomass
Production. Water 2022, 14, 749. [CrossRef]
25. García-Martínez, J.B.; Contreras-Ropero, J.E.; Urbina-Suarez, N.A.; López-Barrera, G.L.; Barajas-Solano, A.F.; Kafarov, V.; Barajas-
Ferreira, C.; Ibarra-Mojica, D.M.; Zuorro, A. A Simulation Analysis of a Microalgal-Production Plant for the Transformation of
Inland-Fisheries Wastewater in Sustainable Feed. Water 2022, 14, 250. [CrossRef]
26. Aragaw, T.A.; Asmare, A.M. Phycoremediation of Textile Wastewater Using Indigenous Microalgae. Water Pract. Technol. 2018, 13,
274–284. [CrossRef]
ChemEngineering 2023, 7, 90 17 of 18

27. Daneshvar, N.; Ayazloo, M.; Khataee, A.R.; Pourhassan, M. Biological Decolorization of Dye Solution Containing Malachite
Green by Microalgae Cosmarium sp. Bioresour. Technol. 2007, 98, 1176–1182. [CrossRef]
28. Dhaouefi, Z.; Toledo-Cervantes, A.; García, D.; Bedoui, A.; Ghedira, K.; Chekir-Ghedira, L.; Muñoz, R. Assessing Textile
Wastewater Treatment in an Anoxic-Aerobic Photobioreactor and the Potential of the Treated Water for Irrigation. Algal Res. 2018,
29, 170–178. [CrossRef]
29. Nagaraj, S.; Sagaya, J.P.J.; Anand, J.; Malairaj, S.; Lakshmaiah, B.; Sathya, R.; MubarakAli, D. A Cyanobacterium Treated Textile
Wastewater for the Plant Growth Enhancement: Experimental Study. Appl. Biochem. Biotechnol. 2022. [CrossRef]
30. Sharif, A. Microbial Degradation of Textile Industry Effluents: A Review. Pure Appl. Biol. 2020, 9, 2361–2382. [CrossRef]
31. Al-Tohamy, R.; Ali, S.S.; Li, F.; Okasha, K.M.; Mahmoud, Y.A.G.; Elsamahy, T.; Jiao, H.; Fu, Y.; Sun, J. A Critical Review on the
Treatment of Dye-Containing Wastewater: Ecotoxicological and Health Concerns of Textile Dyes and Possible Remediation
Approaches for Environmental Safety. Ecotoxicol. Environ. Saf. 2022, 231, 113160. [CrossRef] [PubMed]
32. Lipps, W.C.; Braun-Howland, E.B.; Baxter, T.E. Standard Methods for the Examination of Water and Wastewater, 23rd ed.; American
Public Health Association: Washington, DC, USA, 2017; ISBN 0875532993.
33. Urbina-Suarez, N.A.; Ayala-González, D.D.; Rivera-Amaya, J.D.; Barajas-Solano, A.F.; Machuca-Martínez, F. Evaluation of the
Light/Dark Cycle and Concentration of Tannery Wastewater in the Production of Biomass and Metabolites of Industrial Interest
from Microalgae and Cyanobacteria. Water 2022, 14, 346. [CrossRef]
34. Moheimani, N.R.; Borowitzka, M.A.; Isdepsky, A.; Sing, S.F. Standard Methods for Measuring Growth of Algae and Their Composition
BT—Algae for Biofuels and Energy; Borowitzka, M.A., Moheimani, N.R., Eds.; Springer: Dordrecht, The Netherlands, 2013;
pp. 265–284, ISBN 978-94-007-5479-9.
35. Moheimani, N.R.; Webb, J.P.; Borowitzka, M.A. Bioremediation and Other Potential Applications of Coccolithophorid Algae: A
Review. Algal Res. 2012, 1, 120–133. [CrossRef]
36. Urbina-Suarez, N.A.; Rivera-Caicedo, C.; González-Delgado, Á.D.; Barajas-Solano, A.F.; Machuca-Martínez, F. Bicarbonate-
Hydrogen Peroxide System for Treating Dyeing Wastewater: Degradation of Organic Pollutants and Color Removal. Toxics 2023,
11, 366. [CrossRef] [PubMed]
37. Slavov, A.K. General Characteristics and Treatment Possibilities of Dairy Wastewater—A Review. Food Technol. Biotechnol. 2017,
55, 14. [CrossRef] [PubMed]
38. Urbina-Suarez, N.A.; Barajas-Solano, A.F.; Zuorro, A.; Machuca, F. Advanced Oxidation Processes with Uv-H2 O2 for Nitrification
and Decolorization of Dyehouse Wastewater. Chem. Eng. Trans. 2022, 95, 235–240. [CrossRef]
39. Silva, L.G.M.; Moreira, F.C.; Cechinel, M.A.P.; Mazur, L.P.; de Souza, A.A.U.; Souza, S.M.A.G.U.; Boaventura, R.A.R.; Vilar, V.J.P.
Integration of Fenton’s Reaction Based Processes and Cation Exchange Processes in Textile Wastewater Treatment as a Strategy
for Water Reuse. J. Environ. Manag. 2020, 272, 111082. [CrossRef]
40. Urbina-Suárez, N.A. Different Effect of Nitrogen Sources in Autotrophic and Mixotrophic Culture of Scenedesmus sp. for Biomass
and Carotenoids Production Using Acidic Coal Mine Drainage Effluents. Ing. Y Compet. 2022, 24. [CrossRef]
41. Esther Baby, J.; Jaambavi, I.; Rajeswari, G.; Akshaya, T. Optimization Removal of Colour and Organic Solid Pollutants from
Textile Industry Wastewater by Electrocoagulation. Mater. Today Proc. 2021. [CrossRef]
42. Subashini, P.S.; Rajiv, P. An Investigation of Textile Wastewater Treatment Using Chlorella Vulgaris. Orient. J. Chem. 2018, 34,
2517–2524. [CrossRef]
43. Hussain, Z.; Arslan, M.; Shacir, G.; Malik, M.H.; Mohsin, M.; Iqbal, S.; Afzal, M. Remediation of Textile Bleaching Effluent by
Bacterial Augmented Horizontal Flow and Vertical Flow Constructed Wetlands: A Comparison at Pilot Scale. Sci. Total Environ.
2019, 685, 370–379. [CrossRef] [PubMed]
44. Roy, M.; Saha, R. Dyes and Their Removal Technologies from Wastewater: A Critical Review. Intell. Environ. Data Monit. Pollut.
Manag. 2021, 127–160. [CrossRef]
45. Yao, J.; Mei, Y.; Xia, G.; Lu, Y.; Xu, D.; Sun, N.; Wang, J.; Chen, J. Process Optimization of Electrochemical Oxidation of Ammonia
to Nitrogen for Actual Dyeing Wastewater Treatment. Int. J. Environ. Res. Public Health 2019, 16, 2931. [CrossRef] [PubMed]
46. de Carvalho, J.C.; Borghetti, I.A.; Cartas, L.C.; Woiciechowski, A.L.; Soccol, V.T.; Soccol, C.R. Biorefinery Integration of Microalgae
Production into Cassava Processing Industry: Potential and Perspectives. Bioresour. Technol. 2018, 247, 1165–1172. [CrossRef]
[PubMed]
47. Gita, S.; Shukla, S.P.; Saharan, N.; Prakash, C.; Deshmukhe, G. Toxic Effects of Selected Textile Dyes on Elemental Composition,
Photosynthetic Pigments, Protein Content and Growth of a Freshwater Chlorophycean Alga Chlorella Vulgaris. Bull. Environ.
Contam. Toxicol. 2019, 102, 795–801. [CrossRef] [PubMed]
48. Javed, F.; Rashid, N.; Fazal, T.; Hafeez, A.; Rehman, F. Integration of Real Industrial Wastewater Streams to Enhance Chlorella
Vulgaris Growth: Nutrient Sequestration and Biomass Production. Water Air Soil Pollut. 2023, 234, 1–11. [CrossRef]
49. El-Sheekh, M.M.; El-Shanshoury, A.R.; Abou-El-Souod, G.W.; Gharieb, D.Y.; El Shafay, S.M. Decolorization of Dyestuffs by Some
Species of Green Algae and Cyanobacteria and Its Consortium. Int. J. Environ. Sci. Technol. 2021, 18, 3895–3906. [CrossRef]
50. Shetty, K.; Krishnakumar, G. Algal and Cyanobacterial Biomass as Potential Dye Biodecolorizing Material: A Review. Biotechnol.
Lett. 2020, 42, 2467–2488. [CrossRef]
51. Dellamatrice, P.M.; Silva-Stenico, M.E.; de Moraes, L.A.B.; Fiore, M.F.; Monteiro, R.T.R. Degradation of Textile Dyes by Cyanobac-
teria. Braz. J. Microbiol. 2017, 48, 25. [CrossRef]
ChemEngineering 2023, 7, 90 18 of 18

52. Kumar, A.; Bera, S. Revisiting Nitrogen Utilization in Algae: A Review on the Process of Regulation and Assimilation. Bioresour.
Technol. Rep. 2020, 12, 100584. [CrossRef]
53. Cuellar-Bermudez, S.P.; Aleman-Nava, G.S.; Chandra, R.; Garcia-Perez, J.S.; Contreras-Angulo, J.R.; Markou, G.; Muylaert, K.;
Rittmann, B.E.; Parra-Saldivar, R. Nutrients Utilization and Contaminants Removal. A Review of Two Approaches of Algae and
Cyanobacteria in Wastewater. Algal Res. 2017, 24, 438–449. [CrossRef]
54. Fazal, T.; Rehman, M.S.U.; Javed, F.; Akhtar, M.; Mushtaq, A.; Hafeez, A.; Alaud Din, A.; Iqbal, J.; Rashid, N.; Rehman, F.
Integrating Bioremediation of Textile Wastewater with Biodiesel Production Using Microalgae (Chlorella vulgaris). Chemosphere
2021, 281, 130758. [CrossRef] [PubMed]
55. Gonçalves, A.L. The Use of Microalgae and Cyanobacteria in the Improvement of Agricultural Practices: A Review on Their
Biofertilising, Biostimulating and Biopesticide Roles. Appl. Sci. 2021, 11, 871. [CrossRef]
56. Touliabah, H.E.S.; El-Sheekh, M.M.; Ismail, M.M.; El-Kassas, H. A Review of Microalgae- and Cyanobacteria-Based Biodegradation
of Organic Pollutants. Molecules 2022, 27, 1141. [CrossRef] [PubMed]
57. Tamil Selvan, S.; Dakshinamoorthi, B.M.; Chandrasekaran, R.; Muthusamy, S.; Ramamurthy, D.; Balasundaram, S. Integrating
Eco-Technological Approach for Textile Dye Effluent Treatment and Carbon Dioxide Capturing from Unicellular Microalga
Chlorella Vulgaris RDS03: A Synergistic Method. Int. J. Phytoremediat. 2023, 25, 466–482. [CrossRef] [PubMed]
58. Selvan, B.K.; Pandiyan, R.; Vaishnavi, M.; Das, S.; Thirunavoukkarasu, M. Ameliorative Biodegradation of Hazardous Textile
Industrial Wastewater Dyes by Potential Microalgal sp. Biomass Convers. Biorefinery 2022, 1–12. [CrossRef]
59. Arutselvan, C.; Narchonai, G.; Pugazhendhi, A.; kumar Seenivasan, H.; LewisOscar, F.; Thajuddin, N. Phycoremediation of
Textile and Tannery Industrial Effluents Using Microalgae and Their Consortium for Biodiesel Production. J. Clean. Prod. 2022,
367, 133100. [CrossRef]
60. Ding, T.; Yang, M.; Zhang, J.; Yang, B.; Lin, K.; Li, J.; Gan, J. Toxicity, Degradation and Metabolic Fate of Ibuprofen on Freshwater
Diatom Navicula sp. J. Hazard. Mater. 2017, 330, 127–134. [CrossRef]

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