Environmental Technology

ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: https://www.tandfonline.com/loi/tent20

Harvesting microalgae using ozone-air flotation

for recovery of biomass, lipids, carbohydrates, and
proteins.

María Teresa Valeriano González, María Teresa Orta Ledesma, Sharon

Belinda Velásquez Orta & Ignacio Monje Ramírez

To cite this article: María Teresa Valeriano González, María Teresa Orta Ledesma, Sharon
Belinda Velásquez Orta & Ignacio Monje Ramírez (2020): Harvesting microalgae using ozone-air
flotation for recovery of biomass, lipids, carbohydrates, and proteins., Environmental Technology,
DOI: 10.1080/09593330.2020.1725144

To link to this article: https://doi.org/10.1080/09593330.2020.1725144

Accepted author version posted online: 28

Feb 2020.

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Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group
Journal: Environmental Technology
DOI: 10.1080/09593330.2020.1725144

Harvesting microalgae using ozone-air flotation for recovery of biomass, lipids, carbohydrates, and
proteins.

María Teresa Valeriano Gonzáleza, *María Teresa Orta Ledesmaa, Sharon Belinda Velásquez Ortab,
Ignacio Monje Ramíreza
a
Instituto de Ingeniería, Coordinación de Ingeniería Ambiental, Universidad Nacional Autónoma de
México, Ciudad Universitaria, CP 04510, CDMX, Mexico.
b
School of Engineering, Newcastle University, Newcastle upon Tyne, NE1 7RU England, UK

*Corresponding author; email: tol@pumas.iingen.unam.mx

Abstract

The objective of this research was to study a novel ozone-air flotation microalgae harvesting method
and evaluate its effect on the recovery of biomass and biocomponents (lipids, carbohydrates,
proteins). Best processing conditions were established using a response surface methodology (RSM).
Microalgae separation and biocomponent recovery were evaluated according to changes in gas
concentration (13, 18 and 25 mgO3/L), ozone dose (0.04, 0.09 and 0.16 mg O3/mg biomass) and
airflow rate (0.5, 1.0 and 1.5 L/min). More than 95% of the biomass was recovered from wastewater
at an ozone-air combination of 0.09 mgO3/mg biomass and 1.5 L air/min. Using ozone-air represented
a reduction of 59% in the ozone dose compared to the flotation process solely using ozone (0.22
mgO3/mg biomass). In addition, there was an improved yield in the recovery of all microalgae
biocomponents. A maximum yield of 0.18 mg lipids/mg biomass was achieved at: 0.16 mg O3/mg
biomass, 25 mg gas O3/L and 1.5 L air/min. In conclusion, combining the use of ozone-air for
separation of microalgae reduces ozone requirement and enhances lipids and proteins post-extraction.

1
Keywords: Air-flotation, microalgae biocompounds, biomass separation, microalgae FAMEs

1. Introduction

In recent years, microalgae has emerged as a renewable feedstock for the production of a range of
products in the agricultural, paint and biofuel industries. The enormous potential of microalgal
biomass as a raw material is currently constrained by engineering limitations and challenges. These
limitations include (1) high water demand in the cultivation of microalgae, (2) high nutrient
consumption, and (3) low recovery efficiency. The low recovery efficiency is associated with the
large energy requirements for harvesting and drying biomass (Weschler, et al., 2014). As an
illustration, it is estimated that the biomass harvesting process represents 20-30% of total biofuel
production costs (Rawat, et al., 2011).

On an industrial level, biomass processing is only cost effective at a minimum concentration of 300-
400 g/L (which represents 30-40% solids) (Coward, et al., 2013). Cultivation only delivers low
biomass concentrations of 0.3-5 g/L (0.03-0.5% solids) (Singh and Patidar, 2018; Coward, et al.,
2013). For these reasons, the design of a harvesting system that favors a high microalgal concentration
is essential to obtain any value-added product. Currently, several harvesting methods are available,
including sedimentation, centrifugation, coagulation-flocculation, flotation and ozone flotation
(Alves, et al., 2018; Orta Ledesma, et al., 2018; Barros, et al., 2015; Christenson, 2011).

2
Ozone has been used in water treatment to facilitate particle destabilization and agglomeration. Ozone
causes desorption of the organic coats present in the stabilized particles and reduces the electrostatic
barrier (Jekel, 1994; Reckhow et al., 1986). Similar effects have been reported during microalgae
flotation using ozone (Cheng et al. 2011; Cheng et al. 2010). Nguyen et al., 2013 reported an increase
in the zeta potential of Chlorella sp. from -18.1 to -14.6 mV when using 34 mg O3/L, 5 min ozone as
a pretreatment for the separation of microalgae. Similarly, Nguyen et al., 2013 obtained recoveries
>90% of the algae achieved with 40 mg/L of N-cetyl-N-N-N-trimethyl ammonium bromide and 30
min of ozone. Recently, Alves et al. (2019) observed an increase in zeta potential from -12.1 to -6.6
mV during ozone flotation of Scenedesmus sp. grown in wastewater. Valeriano-Gonzalez et al., 2016
showed that, during ozone flotation, proteins are released that reduce the water surface tension (65.7
to 42.7 mN/m). At a specified critical micelle concentration (CMC: 550 ±17 mg/L), foam is produced
which separates microalgae from the aqueous medium (Valeriano-Gonzalez et al., 2016). In their
study, the C-phycocyanin beta chain was identified as one of the major proteins released during the
ozone flotation process.

Ozone flotation has recently garnered greater interest as a method of harvesting microalgal biomass
and as an alternative for the recovery of biomolecules of commercial interest (lipids, carbohydrates
and proteins). The oxidative properties of ozone provide additional advantages. Flotation conditions
and results for different types of microalgae, culture concentrations and biomolecule composition are
summarized in Table 1. In most cases, ozone is used as a dispersion gas, with doses ranging from
0.005 to 0.52 mgO3/L, depending on the origin of the culture medium (synthetic or wastewater) and
the initial concentration of microalgae. Greater biomass harvesting efficiencies are obtained for
microalgae grown in synthetic media (95-98%) compared to mixed cultures of microalgae grown in
wastewater medium (62-88.5%). A phenomenon that has been consistently reported is that ozone
improves lipid extraction from harvested microalgae, by 2 to 4 times the initial content (Valeriano-
Gonzalez et al., 2016; Velásquez-Orta et al. 2014). Similarly, ozone decreases the degree of
unsaturation of fatty acids (FAMEs) obtained by the transesterification of biomass lipids floated with
ozone.

The objective of this work was to study a novel microalgae ozone-air flotation harvesting method and
to evaluate its effect on microalgal recovery, microalgal lipids, carbohydrates (CHOs), total proteins
(TP) and fatty acid methyl ester (FAMEs) profiling. We selected Scenedesmus obliquus microalgae
as they have been found in the treated wastewater of Mexico City (Valeriano et al., 2016), and
have been cultivated for wastewater treatment (Ferreira et al., 2017). Similarly, their good
biocompound distribution on lipid (12-16%), carbohydrate (30-33%) and protein contents was
also considered (Hernández-García et al., 2019).

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Table 1 Case studies using ozone flotation as a harvesting method.
Initial culture of microalgae Ozone dose
Harvested biomass
Microalgae Biomass Bio-components (mgO3/mg Biomolecules Reference
results
concentration concentration (%, w/w ) Biomass)
- Lipids: 55%
Lipid: 31%
0.005-0.03 - 98% as turbidity -Proteins and polysaccharides were released into Cheng et al.
*C vulgaris CHOs: 8%
600-700 NTU - Agglomerated cells (50µm) the aqueous medium 2010
Protein: 28%
- FAMEs C16:0 : increase from 31% to 55%
- 95% as turbidity
Hydrophobic protein and polysaccharides released Cheng et al.
*S obliquus FSP-3 Lipid: 30-50% 0.2-0.5 - Agglomeration of cells (10-
1.61 g TSS/L during ozone flotation. 2011
50µm)
- Increase in lipid extraction of 14 – 26% compared
** Mixed culture 0.36 g TSS/L Lipid: 20% to centrifugation Komolafe et
0.52
- Increase in saturated FAMEs and decrease in al. 2014
**Desmodesmus sp. 0.58 g TSS/L Lipid: 12% polyunsaturated FAMEs

- Yield: 79.6% as TSS Velásquez-

- Increase in lipid extraction by 12%
** Mixed culture 0.42 g TSS/L Lipid: 6% 0.12 – 0.23 - Harvested concentration: Orta et al.
- Increase in saturated FAMEs
20.2 g/L (2% solid). 2014

- Increase in lipid extraction by 16% compared to

- Yield: 75% as TSS Valeriano-
** Mixed microalgae centrifugation
0.4 g TSS/L Lipid: 8% 0.047 - 0.14 - Harvested concentration: Gonzalez et al.
consortium - Beta C-Phycocyanin was the main protein
20.2g/L (2% solid). 2016
identified released during ozone flotation

- Increased extraction of palmitic acid

Orta Ledesma
**Scenedesmus sp. 0.05 - Yield: 69% as TSS - Ozone flotation increased saturated FAMEs and
- et al. 2017
0.758 g TSS/L decreases polyunsaturated FAMEs.

- Yield: 62% as TSS

- Lipid: 32%
Lipid: 3-8% - Evaluation of the effluent Alves et al.
**Scenedesmus sp. 0.8-1.2 g TSS/L 0.16 - CHOs: 33%
CHOs: 6-44% quality of the flotation ozone 2018
- Protein: 58%
process
Alves et al.
**Scenedesmus sp. 0.5 g TSS/L --- 0.16 -Yield: 88.5% as TSS - Protein: 40%
2019
*: Grown in culture medium; **: Grown in wastewater medium:

4
 2. Methodology

2.1 Microalgae

Experiments were carried out on cultures of microalgae, where Scenedesmus obliquus dominated,
with 80% of the overall concentration. Microalgae cultivations were made using wastewater in PET
bioreactors with a capacity of 10L. The cultivations were carried out in batch processes, with a
photoperiod of light: darkness of 12:12, using LED lamps. The cultivation period was 10-15 days
until a microalgae concentration of 750 mg/L was achieved, measured as total suspended solids
(TSS). TSS were measured according to standard methods (APHA-AWWA-WPCF, 1992).

2.2 Harvesting by ozone-air flotation

Ozone-air flotation harvesting tests were carried out in a skimmer-type reactor. This constituted a
glass column, a foam collector in the upper part, and a glass diffuser (10-15μm pore-sizes) at the
bottom of the column. Ozone-air flotation laboratory-scale experiments used a Labo 76 ozone
generator (Emery Trailigaz, USA) with a production capacity of 19 g O3/h (Figure 1). Ozone and air
were injected through a pore diffuser. The gases were injected separately, i.e. first ozone and then air.
Ozone gas was injected at a flow rate of 0.6 L/min, and the ozone concentration was determined using
the iodometric method (Birdsall et al., 1952). In all experiments, airflow was applied for 4 min.
Ozone-air flotation experiments were conducted in batch processes using 1 L of fresh homogenized
suspensions of microalgae.

Figure 1 Experimental setup for testing the ozone-air flotation with microalgae.

From the ozone-air flotation experiments two products were generated. The first was referred to as
the harvest (concentrate of microalgae and liquid recovered from the top of the reactor), and the
second, flotation effluent (biomass and liquid that remained in the column). Before and after each
experiment, the amount of lipids, carbohydrates, and proteins were measured in the initial biomass of

5
microalgae, in the harvest and in the effluent. Fatty acid methyl ester (FAME) profiling was
determined for the harvested biomass and initial biomass of microalgae.

2.3 Experimental design

The evaluation of the harvesting method was carried out through the determination of the recovered
biomass, lipid (mg lipids/mg biomass), carbohydrates (mg CHOs/mg biomass) and protein yields (mg
proteins/biomass mg). A factorial design for the experiments of 33 was established, taking as
variables: the gas concentration (with three values: 13, 18 and 25 mg O3/L), the ozone dose (with
three values: 0.04, 0.09 and 0.16 mg O3/mg biomass) and the airflow (with three values: 0.5, 1.0 and
1.5 L/min). Each experiment was performed in triplicate, giving 81 tests in total.

Ozone dose (mg O3/mg biomass) was defined as the ozone applied during the harvesting process per
unit of initial biomass. The ozone flotation time (𝒕𝑶𝟑 ) was defined as the time in which the ozone
gas was fed into the skimmer-type reactor, it was calculated according to equation 1.
OD ∗ CB ∗ Vs
𝒕𝑶𝟑 = … equation 1.
CO3 ∗ Q O3

Where, OD is the ozone dose (mg O3/mg biomass), CB is the initial biomass concentration, Vs is the
volume of the sample (L), CO3 is the gas concentration (mg O3/L) and QO3 is the ozone flowrate
(L/min). The percentage of transferred ozone was calculated according to equation 2.
𝑶𝒛𝒐𝒏𝒆 𝑰𝒏 − 𝑶𝒛𝒐𝒏𝒆 𝑶𝒖𝒕
%𝑻𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒓𝒆𝒅 𝒐𝒛𝒐𝒏𝒆 = ( ) ∗ 𝟏𝟎𝟎 … 𝒆𝒒𝒖𝒂𝒕𝒊𝒐𝒏 𝟐.
𝑶𝒛𝒐𝒏𝒆 𝑰𝒏
The yield of recovered biomass was calculated as the difference between the TSS in the harvest and
the TSS not harvested.

Blank experiments were also conducted using ozone flotation as a harvesting method for comparison.
Minitab 17 statistical software was used for the analysis of variance.

2.4 Determination of lipid content

The lipid content was determined by the colorimetric sulfo-phospho-vanillin method (Byreddy et al.,
2016). This was carried out with 1 mL of homogenized suspension of microalgae with a known
concentration. Two milliliters of concentrated sulfuric acid (H2SO4, 98%) were added. This was
followed by incubation in boiling water (100 °C) for 15 min and then immersion in an ice bath for 5
min. Subsequently, 5 mL of phospho-vanillin reagent was added to each sample. They were then
incubated for 15 min at 37°C under constant shaking. The color was allowed to stabilize for a period
of one hour. Canola oil was used for the calibration curve. Finally, generated samples were checked
for absorbance at a wavelength of 530 nm. The lipid content in biomass was determined before
and after the harvest method.

2.5 Determination of carbohydrate content

Carbohydrate concentrations were measured using the colorimetric method for sugars and related
substances (oligosaccharides and polysaccharides) (Dubois, et al., 1956). The technique was carried
out using a solution of 5% phenol (m/m) in distilled water and concentrated H2SO4 (98%). The
procedure was as follows: One milliliter of 5% phenol and 5 ml of H2SO4 (98%) were added to 1 mL

6
of a known homogenized suspension of microalgae, with a known concentration. The mixture was
homogenized and allowed to stand for 10 min. Subsequently, it was placed in a water bath at room
temperature for 15 min. Finally, the absorbance was measured at a wavelength of 490 nm. A
calibration curve was constructed using glucose. The carbohydrate content in biomass was
determined before and after the harvest method.

2.6 Determination of proteins content

The protein content was quantified using the Biuret method (Merck 1103070500), according to the
manufacturer’s specifications. Proteins were extracted from the biomass using the alkali method
(Safi, et al., 2014). This consisted of adding a solution of sodium hydroxide (NaOH) at pH 12 to 1 g
of dry microalgal biomass. The sample was then heated at 40°C with stirring for one hour. The
proteins were recovered from the aqueous phase by centrifugation at 5000 g for 10 min. The protein
content in biomass was determined before and after the harvest method.

2.7 FAME profiling

FAME profiling was carried out after microalgae biomass lipid extraction followed by
transesterification. Lipid extraction was carried out with chloroform: methanol (2:1, v/v), after
samples were vacuum filtered using a Whatman glass microfiber filter paper. Finally, liquid-liquid
extraction was achieved by adding a weak salt solution of potassium chloride (0.88% KCl) at 25% of
the starting volume. Solutions were mixed and two layers were formed. The organic layer was
retained and, subsequently, the organic solvent was evaporated. The lipids extracted were kept for
transesterification. The transesterification reaction was carried out on lipid samples to which 10 ml
of methanol were added. The reaction was catalyzed with H2SO4 at a concentration of 0.04 N. The
reaction was carried out at a temperature of 60 °C, under constant agitation by an orbital
shaker. The transesterification reaction was carried out under solvent: biomass conditions (600:
1) reported to produce lipid conversions of 97% (Velasquez-Orta, et al., 2012).

Consecutively, methanol was evaporated under reduced pressure under an exhaust hood until a final
volume of 2 ml was obtained. Samples were kept at 4 °C, for further qualitative analysis by gas
chromatography coupled to mass spectroscopy (GC-MS). An Agilent Technologies 6809 gas
chromatograph coupled to an Agilent Technologies 5973 mass spectrophotometer was used for
FAME identification. The detection of FAME was achieved using a DB-5ms separation column of
fused silica with a length of 30 m, an internal diameter of 0.25 mm and a film thickness of 0.25 μm.
The carrier gas for analysis was 99.99% pure helium. The GC-MS operating conditions utilized an
injector temperature of 250 °C, with a split type injection; the mass spectrometer was programmed at
a temperature of 280 °C. The oven temperature program was as follows: initial temperature 85 °C (5
min) with a ramp of 7 °C/min, until 265 °C (10 min) was reached.

To perform the qualitative analysis of the FAMEs, the spectrophotometer was operated in SCAN
mode at 50-550m/z. In order to corroborate operating conditions and the results obtained, the peaks
(FAMEs) were identified by comparing the retention time with a known standard of FAME mixture
C8-C24 (Sigma Aldrich, 18918-1AMP, USA). Prior to injecting a 10 µL sample, 10 µL of methanol
were injected to clean the column. On the other hand, in order to establish the percentage of

7
presence of each FAME, the chromatographic peaks areas were considered. Hewlett Packard
ChemStation (B.02.05) was used to integrate the area under the FAMEs peaks. The percentage
of each FAME was calculated using the total area of FAME.

3.0 Results and discussion

3.1 Harvesting by ozone-air flotation

Experimental results demonstrated that an ozone-air flotation system can be used for microalgal
harvesting. The ozone-air flotation system produced an increase in the biomass recovery as TSS of
up to 99% (Table 2). These results can be compared with yields obtained by conventional ozone
flotation (Table 1) (88.5%, Alves, et al., 2019), centrifugation (95%, Singh and Patidar, 2018) or
coagulation-flocculation (98.9%, Reyes and Labra, 2016).

Table 2 Harvested biomass yield against the evaluated factors, AF: Air flow (L/min), GC: gas
concentration (mg O3/L) and OD: ozone dose (mg O3/mg biomass).

Variable Biomass recovery (%)

CO3 (mg O3/L) 13 18 25
AF (L/min) 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5
OD : 0.04 mg O3/mg
64 ± 7 66 ± 2 60 ± 5 87 ± 5 89 ± 4 90 ± 3 74 ± 4 75 ± 9 75 ± 1
Biomass
OD : 0.09 mg O3/mg
64 ± 6 83 ± 1 82 ± 1 87 ± 3 90 ± 2 94 ± 2 53 ± 3 94 ± 6 78 ± 0
Biomass
OD : 0.16 mg O3/mg
79 ± 7 84 ± 4 81 ± 1 87 ± 0 98 ± 1 99 ± 1 70 ± 13 77 ± 6 85 ± 7
Biomass

Table 3 Experimental significance of tested variables in ozone-air flotation.

Variables F-Ratio P-Value

A: Ozone dose 4.73 0.0352
B: Gas concentration 0.05 0.8184
C: Airflow 12.15 0.0011
AB 1.75 0.1926
AC 16.94 0.0002
BC 3.46 0.0698

8
Statistical analysis (ANOVA) (Table 3) showed that, of the variables tested, those that influenced
biomass recovery, in order of significance, were: interaction ozone dose – airflow (p-value =0.0002),
airflow (p-value =0.0011) and ozone dose (p-value =0.0352), respectively. Figure 2 shows the
results of changing ozone dose and concentration at selected levels, keeping airflow constant at
the best found condition of 1.5 L/min. Yields of 95-100% of biomass harvest were achieved through
two combinations of ozone dose and gas concentration: 1) 0.09 mgO3/mg biomass and 18 mg O3/L;
and 2) 0.16 mgO3/mg biomass and 19 mgO3 /L (Figure 2). The best conditions gave an average
microalga concentrate of 21 ± 5 g/L (2.1%w solids), 28 times higher than the initial concentration.

Figure 2 Response surface plot for the harvesting of biomass as a function of gas concentration and
ozone dose.

The surface model (Figure 2), showed that flotation with ozone-air was able to produce
harvested biomass yields between 70-100%. The process allowed a high ozone transfer,
achieving an efficiency of 80%. This means that 80% of the ozone supplied was transferred to
the aqueous medium and, used as an oxidant.

There was no significant difference between ozone-air results and blank experiments, using only
ozone (p-value= 2.2). However, a relevant difference is that using ozone alone required a higher dose
(0.22 mg of O3/ mg of biomass) compared to ozone-air flotation (0.09 mg of O3/mg of biomass). This
showed that flotation with ozone-air reduced the dose of ozone by 59%, and achieved the same yield
of harvested biomass. During ozone-air flotation, ozone is only used for pretreatment, to lyse
microalgae and release surfactant proteins. Surfactant proteins are responsible for carrying
out foam formation and biomass recovery (Valeriano, et al., 2016). After, air flotation was used
to finally recover microalgal biomass.

Table 5 shows that ozone-air flotation requires 6.25 min of ozone, compared to 15.3 min of
conventional ozone flotation (calculation made for 0.22 mg O3/mg Biomass at CO3:18 mg O3/L
and QO3: 0.6L/min. Nava (2015) calculated that ozone flotation had an energy consumption of
0.8 kWh /Ton biomass. Ozone-air flotation would require half the energy (0.475 kWh/Ton

9
biomass). This is because the use of ozone is energy demanding, for instance in the ozone-air
flotation process, it is responsible for 93% of the energy consumed.

3.2 Effect of ozone-air flotation on biocomponents recovery

Other compounds of commercial interest that are contained in the microalgal biomass include long
chain fatty acids, carbohydrates and proteins. Harvesting methods must be able to recover them
efficiently. For ozone flotation, it has been reported that an increase in ozone concentration favors
biomass harvest, but not lipid yield (Valeriano-Gonzalez et al., 2016). The oxidative cell damage
triggered by ozone is responsible for algal cell lysis and the release of biopolymers in the aqueous
medium, where they may be oxidized or remain unrecoverable during ozone flotation.

In this study, the effect of ozone-air flotation on the recovery of lipids, carbohydrates and proteins
was evaluated and compared with the yield of harvested biomass. Statistical analysis (Table 4)
showed that lipid recovery was affected by: gas concentration (p-value: 0.0008), airflow (p-value,
0.0444) and their interaction (p-value: 0.0018). On the other hand, the recovery of carbohydrates
(CHOs) was affected by the gas concentration and airflow interaction (p-value: 0.0379). Finally, the
recovery of proteins was significantly affected by gas concentration (p-value: 0.0000) and airflow-
ozone dose interaction (p-value: 0.0276).

Table 4 Experimental significance of the tested factors: ozone dose, ozone concentration and airflow
for the ozone-air flotation process, in the recovery of mg lipids/mg biomass, mg CHOs/mg biomass,
mg TP/mg biomass. The significance level was 95%.

mg Lipids/mg Biomass mg CHOs/mg Biomass mg TP/mg Biomass

Variable
f-Ratio p-Value f-Ratio p-Value f-Ratio p-Value
A: Ozone dose 1.99 0.1659 0.25 0.6188 0.33 0.5675
B: Gas concentration 13.00 0.0008 0.91 0.3460 43.94 0.0000
C: Airflow 4.29 0.0444 0.85 0.3615 0.05 0.8282
AB 11.01 0.0018 1.84 0.1816 0.90 0.3479
AC 1.29 0.2627 0.10 0.7578 5.20 0.0276
BC 0.70 0.4060 4.59 0.0379 3.42 0.0712

The maximum recovery of lipids, carbohydrates and proteins differed within the conditions tested.
This can be attributed to their location within the microalgal cell, their reactivity with ozone and their
ability to be separated from the aqueous medium by flotation. Figure 3 shows the yields of recovered
biocomponents (mg X/mg biomass) for different ozone-air flotation conditions. In the case of lipids,
an increase in the ozone dose (OD) and gas concentration (GC) improve recovery yields.

10
 0.5 Lipids CHO´s Protein

0.4
mg X/mg Biomass

0.3

0.2

0.1

0
AF 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5
GC 13 18 25 13 18 25 13 18 25
OD 0.04 0.09 0.16

Figure 3 Yield of mg X/mg Biomass (X: lipids, CHOs and proteins) in the biomass harvested against
the evaluated factors: AF (airflow); GC (gas concentration) and OD (ozone dose). Biocomponents
extracted from not treated microalgae: 0.11 ± 0.03 mg lipids/mg biomass, 0.19 ± 0.04 mg CHOs/mg
biomass and 0.38 ± 0.10 mg total protein/mg biomass.

Another factor to take into consideration is the ozonation time, which is related to ozone dose and gas
concentration. For the conditions evaluated, ozonation time varied within a range of 2 to 15 min,
according to equation 1. The highest lipid yields (>0.2 mg lipid/mg biomass) were obtained at short
ozone flotation time (4 min), which was achieved at high gas concentration (25 mgO3/L) and high
ozone dose (0.16 mgO3/L) (Figure 4). The effect of ozone concentration (p-value: 0.0008) can be
observed in the subsequent recovery of microalgae lipids (Figure 4). As can be seen, a low ozone
concentration did not produce adequate lysis of microalgae, thus unabling a high yield of
extracted lipids. On the other hand, an increase in ozonation time (>6 min) at different gas
concentrations reduced the yield of lipids present in the biomass, which can be attributed to the
oxidizing effect of ozone (Kumari et al. 2013). It has been reported elsewhere that ozone reacts with
the unsaturated bonds of mono and polyunsaturated fatty acids, forming saturated and smaller fatty
acids (Cunha et al. 2011), with ketones, aldehydes, hydrocarbons and malondialdehyde formed as by-
products (Roshchina and Roshchina, 2003).

11
Figure 4 Effect of gas concentration and ozonation time on lipid recovery.

Lipid content in filtered samples of the flotation effluent was quantified to determine the release of
lipids from the cells to the aqueous phase during ozone-air flotation. The initial concentration of lipids
in the aqueous medium, prior to flotation, was 14.5 ± 2.26 mg lipid/L, in the harvested medium this
was 28 ± 3.76 mg/L and in the effluent after flotation they were not detected. These results indicate
that a small amount of lipid was released from the cells by the oxidative effect on the microalgae, but
this was recovered in the harvest. Hence, there was no loss of lipids due to the harvesting process.

With respect to carbohydrates, it was observed that for the majority of the combinations of ozone
doses and gas concentrations tested, the recovery efficiency increased with airflow (Figure 3).
Optimal results were obtained with ozone doses of 0.09 mg/mg of biomass; gas concentration 13
mg/L; air flow 1.5 L/min. Unlike lipids, and depending on the conditions used, carbohydrates may be
released in greater quantities in the aqueous phase during ozone-air flotation. This can be attributed
to the fact that carbohydrates are the main component of the microalgae cell wall (Chen, et al., 2013).
Therefore, they are more likely to be exposed to the action of ozone and are thus more easily released
to the aqueous phase. However, due to their structure, they have low reactivity with ozone, and their
recovery is therefore difficult. Optimal ozone-air flotation conditions for carbohydrate release,
produced 0.14 mg CHOs/mg biomass in the harvested microalgae; and 224.6 mg CHOs/L in the
harvested aqueous phase.

For protein recovery the most significant variable was gas concentration (p-value: 0.0000), and
indirectly ozonation time. All three tested ozone dose and airflow conditions, produced yields >0.4
mg total protein/mg biomass. However, these results were mainly obtained at the highest gas
concentration (25 mgO3/L), an ozone dose of 0.04 mg O3/L and an airflow of 1.5 L/min (Figure 3).
It should be noted that during ozone flotation, a large amount of foam is formed due to the release of
proteins from microalgae cells and the effect of gas dispersion. In fact, this is the mechanism used to
separate and harvest microalgal biomass in the upper collector of the flotation reactor. Similarly to

12
the ozone effect in lipids and carbohydrates, using low gas concentrations increased contact time and
therefore there was more probability for proteins to react with ozone.

Table 5 shows the best operating conditions obtained for ozone-air flotation to achieve the highest
extraction yields of lipids, carbohydrates, or proteins in the biomass. It can be seen that a high biomass
recovery requires a high ozone flotation time compared to the recovery of biocomponents. This can
be attributed to the fact that a long ozone flotation time, lysed microalgae to the point of affecting the
amount of biocomponents recovered.

Table 5 Ozone-air flotation process conditions obtained from the response surface methodology and
statistical analysis for the maximum yield of biomass, lipids, carbohydrates and proteins.
Variables Biomass Lipids CHOs Protein
Ozone dose (mg O3/mg biomass) 0.09 0.16 0.09 0.04
Gas concentration (mg O3/L) 18 25 13 25
Ozone flotation time (min) 6.25 4.0 4.5 4.0
Airflow (L/min) 1.5 1.5 1.5 1.5
Air flotation time (min) 4 4 4 4
Estimated yield (mg/mg biomass) 0.98 0.18 0.14 0.5
Biocomponents extracted from no treated microalgae: 0.11 ± 0.03 mg lipids/mg biomass, 0.19 ± 0.04 mg
CHOs/mg biomass and 0.38 ± 0.10 mg total protein/mg biomass.

3.3 Effect of ozone-air flotation on FAME composition

FAME profiles were obtained after transesterification of the lipids extracted from Scenedesmus
obliquus harvested at the best lipid recovery conditions (Table 6). To compare the effect of ozone,
the lipid profile was also determined in samples of centrifuged biomass (3000 rpm x 10min).
Linolenic (C18:3n3c) and palmitic (C16:0) acids were the most abundant fatty acids produced by
Scenedesmus obliquus microalgae. Palmitic acid (C16: 0) was the most abundant FAME in biomass
harvested by ozone-air flotation. On the other hand, linolenic acid (C18:3n3c) had greater relative
abundance in the biomass centrifuged than in the biomass separated by ozone-air flotation. As
previously reported, during the ozone flotation of microalgae ozone reacts with fatty acids and
reduces the degree of unsaturation (Komolafe et al., 2014; Velazquez-Orta et al., 2014). During the
present work, it was observed that the biomass harvested by the ozone-air flotation method contained
a high percentage of saturated fatty acids. It also produced a decrease in the amount of
polyunsaturated fatty acids such as linolenic acid (C18:3n3c).

In order to explain the effect of ozone on the composition of the lipid profile, it should be noted that
there are neutral or non-polar lipids and polar lipids within the lipid composition of microalgae.
Neutral lipids (mainly triglycerides) are considered important for energy storage in microalgae and
are located as lipid bodies in the cytoplasm. On the other hand, polar lipids (mainly polyunsaturated
fatty acids) are used in the membrane structure of the microalgae (Prabandono and Amin, 2015; Hu,
et al., 2008). In accordance with the location of the lipids in the microalgae, ozone reacts first with
the lipids present in membranes, increasing the degree of saturation of the polar lipids, and allowing
the extraction of neutral lipids. This is corroborated with the increase in saturated fatty acids at the
end of the ozone-air flotation process.

13
Table 6 Fatty acid profile for Scenedesmus obliquus grown in wastewater and harvested by
centrifugation and ozone-air flotation using qualitative GC-FID (in-situ transesterification).
Ozone-air flotation
FAME Type Centrifugation Ozone dose: 0.16 mg O3/mg Biomass
GC: 25 mg O3/L GC: 18 mg O3/L
C14:0 - 0.76 0.46
Myristic
C15:0 - - 0.48
Pentadecanoic
C16:0 22.33 35.89 37.66
Palmitic
C16:1n9c 5.85 9.17 8.00
Palmitoleic
C16:2 0.62 2.74 -
7, 10 hexadecadienoic acid
1.23 2.20 1.34
C16:3
7, 10, 13 hexdecatrienoic acid
- 1.22 -
C17:0
Heptadecanoic acid
0.88 2.91 1.59
C18:0
Stearic
6.55 1.79 9.19
C18:1n9c
Oleic
4.10 13.25 10.44
C18:2n6c
Linoleic
58.43 28.01 27.79
C18:3n3c
Linolenic
- 2.05 1.40
C22:0
Docosanoic acid
Saturated* 23.21% 42.83% 42.29%
Monounsaturated* 12.40% 10.96% 17.48%
Polyunsaturated* 64.39% 46.20% 40.23%
C16-C18* 23.21% 38.80% 39.91%
* Calculation based on the percentage of the FAME of interest (mg) compared to the total FAME (mg).

3. Conclusions

An ozone-air flotation method for microalgae harvesting from wastewater was developed and
evaluated using a surface response methodology. Operating conditions for this microalgae harvesting
system were established through the combination of the use of ozone as a pretreatment followed by

14
flotation with air (ozone-air flotation). Ozone-air flotation produced the same biomass harvesting
efficiency as that obtained by ozone flotation, but with ozone dose savings of up to 59%. Similarly,
an improvement in the recovery performance of some biocomponents, such as lipids and proteins,
was recorded. The ozone dose, gas concentration, airflow and contact time may be selected depending
on the interest in lipid, carbohydrate or protein recovery. Carbohydrates and proteins were released
from the biomass to the liquid phase because of ozone-microalgae interaction. It should be noted that
using an ozone-air system, like ozone alone, improves accessibility to lipids and favors the saturation
of fatty acids. The optimum yields of lipids were obtained with short ozone flotation time (4 min) and
ozone dose (0.16 mgO3/L). An increase in ozonation time (>6 min) and different gas concentrations
reduced the yield of lipids present in the biomass, which can be attributed to the oxidizing effect of
ozone. Comparison of centrifuged and ozonated biomass, showed that the saturated FAME and
quantity of C16-C18 was increased by the ozone–air flotation process.

Acknowledgements

The authors thank MSc. Isaura Yáñez-Noguez for her help in the identification of microalgae. This
work was supported by the CONACYTSENER-Sustainability Energy Sector Fund, project 220704.

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