Environmental Technology & Innovation 35 (2024) 103677

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Simultaneous veterinary antibiotics reduction and biomass

production from pig slurry by microalgae co-immobilization in
moving bed biofilm reactors
Mònica Escolà Casas *, 1, Edward J. Pastor-López 2, Yolanda Rodríguez-Espelta 3,
Víctor Matamoros 4
Department of Environmental Chemistry, IDAEA-CSIC, c/Jordi Girona, 18-26, Barcelona 08034, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: The objective of the study was to assess, for the first time, the effectiveness of using either freely
Pig slurry suspended or co-immobilized microalgae on Moving Bed Biofilm Reactor carriers in removing
Microalgae lincomycin from the liquid fraction of pig slurry, while ensuring the safe production of biomass.
MBBR
To this end, continuous-mode (HRT = 8 d) and batch-mode (10 d) experiments were conducted
Lincomycin
on bench-scale reactors. In the continuous-mode experiment, co-immobilized microalgae
Ammonium
Removal removed 99 % of lincomycin and 94 % of ammonium, whereas the control reactors and free-
microalgae reactors showed significantly lower ammonium removals (69 % and 87 %, respec­
tively). The removal rates for lincomycin and ammonium in co-immobilized microalgae reactors
were higher (0.85 and 0.32 d− 1, respectively) than the removal rates of the free-microalgae re­
actors (0.32 and 0.19 d− 1) and control reactor (0.18 and 0.12 d− 1). Furthermore, the microalgae
reactors were linked with the production of lincomycin transformation products following
ammonium and nitrate removal. In contrast to the control reactors, the suspended biomass of
both free microalgae and co-immobilized microalgae reactors showed no accumulation of
lincomycin. This characteristic makes this biomass particularly appealing in the context of a
circular economy. The study introduces an innovative method for producing biomass from pig
slurry, with the goal of obtaining high-value products while minimizing pollutant content.

1. Introduction

Pig slurry management should necessarily involve effective treatments that are both sustainable for the farmer’s production
business and beneficial for the ecosystem (Martinez-Almela and Barrera, 2005). Currently, pig slurry can be directly spread on soil or
treated by means of different technologies, such as nitrification-denitrification (NDN) treatment or anaerobic digestion (Yuan et al.,
2018). Another option is to separate the pig slurry into liquid and solid fractions, which is normally done to reduce the volumes of

* Corresponding author.
E-mail address: mecqam@cid.csic.es (M. Escolà Casas).
1
ORCID: 0000–0002-1282–5515
2
ORCID: 0000–0002-1685–1518
3
ORCID: 0009–0004-8816–0865
4
ORCID: 0000–0001-9701–4908

https://doi.org/10.1016/j.eti.2024.103677
Received 4 January 2024; Received in revised form 20 April 2024; Accepted 13 May 2024
Available online 15 May 2024
2352-1864/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Escolà Casas et al. Environmental Technology & Innovation 35 (2024) 103677

animal waste and, thus, the associated transportation costs (Hjorth et al., 2010). With this option, the solid fraction is normally
composted, while the liquid fraction is used for agricultural soil fertilization due to its high nitrogen content. Nevertheless, the liquid
fraction contains highly polar veterinary antibiotics such as lincomycin at a concentration up to 130 µg/L (Marti et al., 2020).
Therefore, when farmers use the liquid fraction for fertigation, these compounds can be taken up by crops and pose a risk to human
health (Margenat et al., 2020). Furthermore, veterinary antibiotics can also reach surface water and groundwater, leading to risks for
both ecosystem health and human well-being (Kivits et al., 2018; Mehrtens et al., 2021). To overcome this problem, different strategies
can be applied to treat the liquid fraction, such as straw filtration, microfiltration, NDN, or evapotranspiration, but these technologies
are not often effective for removing veterinary antibiotics and are expensive to build and maintain (Melse and Verdoes, 2005). In
recent years, the use of microalgae to treat pig slurry has also attracted attention (Sánchez-Zurano et al., 2021). For example, Juárez
et al. suggest the complete valorization of the biomass (microalgae) from pig slurry to produce high and medium-value compounds,
applying sequential and selective processes (Juárez et al., 2020).
3−
The liquid fraction of pig slurry is rich in nitrogen (essentially N-NH+4 ) and phosphorus (mainly P-PO4 ), as well as organic matter
and minerals such as calcium and magnesium (Luján-Facundo et al., 2019). Recent studies have demonstrated that this liquid fraction –
previously diluted to avoid N-NH4 toxicity- can be used as a nutrient source for microalgal biomass production, and that it is a good
solution for pig slurry revalorization (Ciardi et al., 2022). A few studies have shown that free and co-immobilized microalgae (together
with other microorganisms) can be used to treat wastewater (Molazadeh et al., 2019), enabling nutrient recovery through the resulting
microalgal biomass. Furthermore, recent results have highlighted the usefulness of microalgae-based technology to remove antibiotics
from wastewater and groundwater (Ferrando and Matamoros, 2020; Xiong et al., 2021). In this sense, immobilization techniques have
proven more efficient in treating wastewater than free-microalgae (suspended-growth) systems (Lau et al., 1997; Solé and Matamoros,
2016; Salman et al., 2022). Among the various microalgae immobilization strategies, recent findings have shown that passive
co-immobilization via microalgal-biofilm growth (which includes microalgae and heterotrophic microorganisms) on different surfaces
is a cheap strategy that can be successfully adapted to different conditions, and which tolerates antimicrobial substances (Mantzorou
and Ververidis, 2019). Moving Bed Biofilm Reactors (MBBRs) are designed for wastewater treatment and use microbial biofilms
(without microalgae) grown on plastic carriers that are continuously stirred to treat wastewater. This approach has been applied to
treat different types of wastewaters (e.g., municipal (di Biase et al., 2019), hospital (Escolà Casas et al., 2015), dairy (Santos et al.,
2020), dyeing (Yang et al., 2020), and refinery industries (Schneider et al., 2011)). Recently, MBBRs were used to co-immobilize
microalgal biofilms, which were proven to remove nitrogen from synthetic wastewater (Wu et al., 2023). However, MBBRs with
co-immobilized microalgal biofilms have never been used for the direct treatment of swine wastewater.
Based on the available knowledge, for the present study, it was hypothesized that microalgae co-immobilized on MBBR carriers
could enhance lincomycin and ammonium removal from pig slurry compared to free microalgae. If this hypothesis is true, microalgal
biofilms grown in MBBRs or on other types of carriers could be a good on-site and cost-effective solution to treat pig slurry.
Furthermore, this treatment would offer a way to recover nutrients and compounds of added value such as pigments or phytoesti­
mulants obtained from microalgae as previous studies have shown (Bellver et al., 2023; Álvarez-González et al., 2023). Thus, this study
aims to assess for the first time how effectively microalgae can eliminate lincomycin, a polar antibiotic, while also providing the
opportunity to produce lincomycin-free biomass. Additionally, it explores the enhanced effectiveness of co-immobilizing microalgae
by employing MBBR carriers.

2. Material and methods

2.1. Reactor set-up

The liquid fraction of pig slurry was originally lincomycine-free and it was obtained from a pig farm located in Catalonia. Such
− 1 − 1 - − 1 3- − 1
fraction had the following composition: NH+ 4 = 3335 mg L , NO3- = 82.2 mg L , NO2 = 9.055 mg L , PO4 = mg L , pH = 7.14,
− 1 − 1
COD = 6330 mg L , and suspended solids = 49.1 mg L . Due to the high ammonium concentration in the original slurry, which it was
found to be toxic to the microalgae, the liquid fraction was diluted by a factor of five, as has previously been done elsewhere (Kwon
et al., 2020; Park et al., 2010). The diluted liquid fraction of the pig slurry was then spiked with lincomycin to achieve a concentration
of 200 µg L− 1, which is a typical concentration range in which this compound can be found in the liquid fraction of pig slurry (Wohde
et al., 2016).
To achieve a microalgae consortium adapted to grow in pig slurry, a 1.5 L cylindrical reactor made of methacrylate containing the
diluted pig slurry and 150 MBBR carriers was inoculated with a previous microalgae-bacteria consortium stock (Rambaldo et al.,
2022). The carriers were continuously mixed by using bottom aeration. The MBBR carriers were AnoxKaldnes™ K5 carriers (Anox­
Kaldnes, Lund, Sweden) and were made of high-density polypropylene (25 mm in diameter each), with a protected surface of 800 m2
m− 3. Microscopic examination showed that most of the microalgae present were colonial and unicellular green algae (Scenedesmus and
Chlorella genera mainly). The reactor was fed with diluted pig slurry in a semi-continuous mode with an HRT of 8 days for 1 month.
This HRT was obtained empirically, by controlling nitrification over time. Afterwards, two experiments were conducted (one in
continuous-operation and the other in batch mode) to assess the effectiveness of free microalgae and co-immobilized microalgae on
MBBR carriers for the attenuation of lincomycin and ammonium.

2.1.1. Continuous-operation experiment

First, a continuous operation experiment was conducted. To this end, one control reactor containing diluted pig slurry, one control
reactor containing diluted pig slurry and MBBR carriers, one reactor containing free microalgae grown in diluted pig slurry without

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M. Escolà Casas et al. Environmental Technology & Innovation 35 (2024) 103677

Fig. 1. Experimental set-up of the continuous-operation and batch-mode experiments.

MBBR carriers, and one reactor containing co-immobilized microalgae on MBBR carriers grown on diluted pig slurry were tested
(Fig. 1). Each reactor contained 250 mL of diluted pig slurry (0.37±0.03 g DW L− 1) or free-microalgae culture grown in diluted pig
slurry (0.82±0.02 g DW L− 1) and, in the reactors with carriers, 42 new MBBR carriers or 42 MBBR carriers grown on the media
inoculated with microalgae (Fig. 1). All reactors were set up in a temperature-controlled growth room at 23 ± 5 ◦ C and lit by fluo­
rescent tubes at a photon flux density of 150 μmol m− 2 s− 1 in a 12 h light/12 h dark cycle (Philips Master TL-D, 36 W/840). Gentle
agitation (ca. 200 rpm) was provided with a magnetic stirrer. All reactors were operated in semi-continuous mode at an HRT of 8 days
by removing and adding 30 mL of diluted pig slurry spiked with lincomycin every day. This was performed for 30 days. After that,
samples from the reactors were collected every second day for 10 days for lincomycin and nutrient analysis (n=5). The last three
sampling days, biomass samples were collected from each reactor by filtration with glass-fiber filters (GF/F). Also, 3 carriers from each
type of MBBR reactor were collected for further biomass analysis. Biomass from filter samples and MBBR carriers was dried at 105 and
50 ◦ C to constant weight. In addition, the amount of lincomycin contained in fresh biomass from the glass-fiber filter was analyzed.

2.1.2. Batch experimental set-up

After the continuous-operation experiment, a batch experiment was performed to test all the previous conditions except for the
control reactors containing only diluted pig slurry (Fig. 1). To obtain the lincomycin degradation curves, three reactors were tested for
each condition. To do so, 100 mL reactors containing 50 mL of new diluted pig slurry and 50 mL of the effluent wastewater from the
corresponding reactors in the continuous mode were used. Afterwards, 13 new MBBR carriers or 13 MBBR carriers grown on the media
inoculated with microalgae were added to the corresponding reactors (Fig. 1). Each reactor was spiked to 200 µg L− 1 of lincomycin.
Gentle agitation was provided with a magnetic stirrer (ca. 200 rpm). As soon as the reactors were set up (within 10 minutes), samples
for t=0 were taken. Additional samples were then taken after 1, 3, 6, and 10 days for the determination of the lincomycin, ammonium,
and nitrate concentration levels.

2.1.3. Transformation products (TPs) determination

To determine the TPs generated by microalgae, two types of reactors were assembled: three reactors containing 80 mL of
microalgae culture grown in diluted pig slurry, 20 mL of filtered pig slurry, and 13 MBBR carriers; and three other reactors containing
only 100 mL of filtered pig slurry. All reactors were spiked to 400 µg L− 1 of lincomycin. Magnetic agitation was applied and left for
1 minute before the first sampling (t=0). Samples were then taken at 6 h, 24 h, 48 h, 72 h, and 5 days. Samples from each reactor type
and sampling time were pooled and filtered with a centrifuge filter (0.22 µm PTFE hydrophilic membrane). Filtrates were directly
injected into a UPLC-QToF for TP determination.

2.2. Analytical methodology

Ammonium and nitrate concentrations in the water were analyzed using Hach Lange Ammonium (LCK305) and Nitrate (LCK339
and LCK340) cell tests on a Hach Lange DR 1900 Portable Spectrophotometer. To determine lincomycin in the aqueous samples, the
samples were filtered with 2 mL centrifuge filters (0.22 µm PTFE hydrophilic membrane), and the filtrate was directly injected and
analyzed by liquid chromatography coupled to mass spectrometry (LC-MS). To measure lincomycin in the suspended biomass of the
corresponding samples, 30 mL of water were filtered through a 0.7 μm, GF/F filters and about 1 g of the fresh biomass retained in the
filter (pig slurry or microalgae) was weighted and extracted following a previously published method (Tadić et al., 2019). Briefly, the
biomass was suspended in 10 mL of methanol, sonicated for 15 minutes, and centrifuged. The supernatant was evaporated to 1 mL and
then reconstituted to 20 mL with ultrapure water. These 20 mL were loaded on SPE cartridges (Strata-X, 100 mg/6 mL) previously
conditioned with 6 mL of methanol and 6 mL of ultrapure water. After loading the sample, the cartridges were washed with 10 mL of
ultrapure water containing 5 % of methanol and then dried. Finally, the cartridges were eluted with 10 mL of ethyl acetate, dried under
a nitrogen stream, and reconstituted in ultrapure water. These extracts were also analyzed by LC-MS.

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Table 1
Lincomycin, ammonium, and nitrate concentrations in the influent water and the reactors operated in continuous mode (HRT of 8 days) and their
corresponding average removal efficiencies. Of the latter, means not sharing a letter are significantly different according to Tukey’s HSD test with a
95 % level of significance.
Reactor type (n=3) Influent concentration [mg L− 1] Reactor concentration (8 d HRT) [mg L− 1] Removal efficiency (%)

Lincomycin
Control (only pig slurry) 253 ± 23 62 ± 12 76 ± 4a
Control MBBR 253 ± 23 31 ± 8 88 ± 3b
Free microalgae 253 ± 23 < LOD 99 ± 1c
MBBR microalgae 253 ± 23 < LOD 99 ± 1c
Ammonium (NH+ 4)
Control (only pig slurry) 583 ± 33 187 ± 2 68 ± 2a
Control MBBR 583 ± 33 172 ± 8 69 ± 1a
Free microalgae 583 ± 33 69 ± 7 87 ± 1b
MBBR microalgae 583 ± 33 33 ± 8 94 ± 2c
Nitrate (NO-3)
Control (only pig slurry) 360 ± 35 912 ± 43 -154 ± 22
Control MBBR 360 ± 35 729 ± 463 -174 ± 43
Free microalgae 360 ± 35 648 ± 413 -518 ± 319
MBBR microalgae 360 ± 35 1973 ± 953 -80 ± 115

The LC-MS equipment consisted of a Waters Acquity Ultra-Performance Liquid Chromatography™ System (Milford, MA, USA)
coupled with a Waters TQ-Detector mass spectrometer (Manchester, UK). The chromatographic separation was conducted on a Kinetex
C18 5 cm×2.1 mm×2.6 µm column (Phenomenex) at 25 ◦ C with a binary gradient elution program using water with 0.1 % formic acid
and acetonitrile with 0.1 % formic acid as mobile phases. Lincomycin was determined with tandem mass spectrometry in multiple-
reaction-monitoring (MRM) mode using two transitions. Ions were generated using electrospray ionization in positive mode
(ESI+). Further details on the analytical methodology and quality parameters are provided in Section 1 of the SM.
To determine lincomycin TP formation, filtered samples were directly injected into a UPLC-QToF (Bruker Impact II) with a
chromatographic separation matching the conditions of the instrument software TargetScreener HR. To this end, a C18 column (Bruker
Intensity Solo; L=100 mm, ID=2.1 mm; particle size=1.8 µm; with a precolumn) was used, operated at 40 ◦ C. The mobile phases
consisted of water/methanol (99:1) and methanol, both with 5 mM of ammonium formate and 0.01 % of formic acid. Electrospray
ionization was used in positive mode, and the mass spectrometry was with data-dependent MS/MS. Once the data were obtained,
MetaboScape® software (Bruker) with the built-in BioTransformer 3.0 solution (Djoumbou-Feunang et al., 2019) was used for the
identification of TPs by predicting different biotic and abiotic reactions of lincomycin and comparing the resulting molecular formulas
with the obtained data.

2.3. Data analysis

Lincomycin, ammonium, and nitrate attenuation efficiencies were calculated from measured influent and effluent reactor con­
centrations of each of the studied reactors (continuous-operation experiment). Lincomycin and ammonium degradation rates (k) were
then calculated from the concentration-decay over time plots resulting from the batch experiment. To this end, normalized concen­
tration data over time were fitted to the one-phase decay equation (Eq. 1) (Schwarzenbach et al., n.d.), with no weighting of the data.
Y = [Y0 ]exp ( − k ∗ X) (1)

Where Y0 (initial normalized concentration) was constrained to be 1. For the ammonium depletion rate, Eq. 1 was fitted up to X=8 d,
because the data after this day changed the trend, most likely due to system starvation (see Fig. 2).
The percentage of the k (for lincomycin and ammonium) from the co-immobilized microalgae on MBBR explained by the diluted
liquid fraction of pig slurry was considered to be the k of the control containing MBBR carriers. Likewise, the percentage of the k due to
the presence of free microalgae and the diluted liquid fraction of pig slurry was considered to be the k of the free-microalgae reactor.
Finally, the removal rate constants (k) were divided by the dry biomass concentration in each reactor (suspended biomass and
attached biomass, where present) to obtain the removal rate constants (k) normalized to the biomass concentration (kbio).

3. Results and discussion

3.1. Continuous-operation experiment

During the operation period, the pH of the all reactors was maintained between 8.4 and 9.0, as they were only being agitated.
Regarding suspended biomass, in the end of the continuous operation, the reactors operated with slurry or with slurry with MBBR
carriers contained 0.40±0.10 g DW L− 1 and 0.51 ± 0.17 g DW L− 1 of suspended solids respectively. Instead, the reactors operated with
free or immobilized microalgae contained 0.72±0.06 g DW L− 1 and 0.55±0.17 of suspended solids correspondingly (not accounting
for the immobilized biomass). This shows that the suspended solids did not vary in the reactors containing only slurry, but they
increased on the reactors containing microalgae. However, the co-immobilized microalgae had reduced suspended solids in

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-
Fig. 2. Lincomycin, ammonium (NH+ 4 ), and nitrate (NO3) concentrations normalized over time during the kinetic assessment study. Curves shown
on the lincomycin graph are the fitted curves following Eq. 1; their parameters can be seen in Table 2. Lincomycin was analyzed for each reactor,
whereas a pool of the three reactors was used to measure ammonium and nitrate concentrations in order to minimize volume changes.

comparison to the free microalgae. This was expected as MBBR reactors are usually producing low suspended solids (Barwal and
Chaudhary, 2014). Accordingly, this trend is also followed by the COD values obtained in the end of the experiment, which were of
1406 mg L− 1and 1413 mg L− 1f or the reactors containing pig slurry or pig slurry with carriers and 1696 mg L− 1 and 1358 mg L− 1 and
or the reactors with free or co-immobilized microalgae respectively. This data indicates that the usage of a reactor with co-immobilized
microalgae produces water with a better quality than the reactor with free microalgae in terms of suspended solids and COD.
The attenuation of lincomycin from pig slurry in the different reactor configurations during continuous operation at an HRT of 8
days ranged from 76 % to 99 % (Table 1). The control reactors showed lower lincomycin removal than the reactors containing
microalgae (76–88 % vs. 99 %). The attenuation of lincomycin in the control reactor containing only diluted pig slurry can be
explained by either photodegradation or biodegradation (Lei and Lai, 2019; Mehrtens et al., 2021). The present results reveal that the
presence of MBBR carriers in the other control reactors aided in the creation of a very thin layer of biofilm that statistically increased
the lincomycin attenuation, from 76 % to 88 %. These removal efficiencies are similar to those found in an NDM system in which the
attenuation of lincomycin from pig slurry ranged from 79% to 99%, but which was operated with a greater HRT (30 days instead of the
8 days used with the MBBR photobioreactor here) (Marti et al., 2020). Additionally, while both microalgae reactors (free and
co-immobilized) showed greater lincomycin attenuation efficiencies (99 %), no differences were detected between them (Table 1).
This might be due to the microalgae reactors’ great efficacy at an HRT of 8 days (99 % removal, on average), which made it impossible
to detect variations. In this case, the batch experiment was useful to check possible degradation-kinetics differences between reactors
(see Section 3.2). The greater effectiveness in removing lincomycin could be linked to a greater nitrifying environment of such reactors.
In fact, this has been observed in a previous study (Zhou et al., 2021) and it is supported by the TPs data, which show the formation of

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Table 2
Kinetic parameters for ammonium and lincomycin obtained from the batch experiment. Ammonium equations and removals were adjusted until t =
8d.
K (d− 1) 1
Kbio (L g−biomass d− 1) t1/2 (d), 95% confidence intervals Correlation coefficient (R2) Removal efficiency (%)

Lincomycin
Control MBBR 0.18 ± 0.03 0.30 3.12–4.76 0.85 74
Free microalgae 0.32 ± 0.06 0.44 1.74–2.70 0.92 100
MBBR microalgae 0.85 ± 0.19 0.58 0.64–1.02 0.93 100
Ammonium (NH4+)
Control MBBR 0.12 0.20 4.34–7.32 0.96 60
Free microalgae 0.19 0.26 2.87–4.55 0.98 83
MBBR microalgae 0.32 0.22 1.31–3.34 0.95 98

lincomycin TPs with an added oxygen (see Section 3.2). Although the great effectiveness of microalgae-based technologies for
removing antibiotics has already been described in wastewater and groundwater (Ferrando and Matamoros, 2020; Leng et al., 2020),
this is the first time it has been proved in pig slurry.
Table 1 also shows the attenuation of ammonium and the formation of nitrate during the continuous operation of the reactors. The
results indicate that, whereas the attenuation of ammonium was about 70 % in the control reactors, the presence of microalgae
enhanced it significantly, to 87 %, and the co-immobilization of microalgae on MBBR carriers achieved even higher removals (94 %).
This can be explained first, by the fact that microalgae have already been found to assimilate ammonium (Salbitani and Carfagna,
2021) and, second, by the presence of nitrifying bacteria (Akizuki et al., 2019). These results are similar to those reported by
Sánchez-Zurano et al. (Sánchez-Zurano et al., 2021), who observed an ammonium removal of 90 % from a diluted pig slurry
(290–300 mg L− 1 N-ammonium) using a photobioreactor operated in continuous mode with an HRT of 5 days. Furthermore, the fact
that the co-immobilization of microalgae and bacteria on MBBR carriers increases ammonium removal is consistent with previous
studies of wastewater treatments, in which the symbiosis between bacteria and microalgae enhanced the attenuation of ammonium
(De-Bashan et al., 2002).
The calculated removal rates for ammonium from the continuous-operation systems (free and co-immobilized microalgae) were of
64 and 69 mg L− 1 d− 1, respectively (initial concentration of 583 mg L− 1). These values are fairly consistent with those reported by
Sánchez-Zurano et al. (Sánchez-Zurano et al., 2021), who observed that at a slightly different initial concentration of ammonium
(300 mg/L), the removal rate ranged from 40 to 55 mg L− 1 d− 1 when microalgae were used. The differences with Sánchez-Zurano et al.
may be due to differences in pig slurry composition or microalgae species. Future studies should thus include such factors in the
assessment of the use of MBBR photobioreactors for nutrient recovery and antibiotic removal from pig slurry. In terms of biomass
efficiency, taking in account the total biomass of the reactors, the ammonium removal rates were of 89 mg L− 1 d− 1 g DW− 1 and
47 mg L− 1 d− 1 g DW− 1 for the free and co-immobilized microalgal reactors. This shows that, per unit of biomass, the co-immobilized
microalgae were about 50 % less efficient in removing ammonia. Nevertheless, the immobilization allowed allocating more biomass in
the reactor in the form of biofilm while achieving lower values of total suspended solids in the aqueous fraction than the free
microalgae reactor.
Although ammonium was removed, nitrate was produced in all reactors. This suggests that the ammonium was removed mainly by
nitrification. The free-microalgae reactor achieved greater nitrate production than the control reactor, which can be explained by the
greater attenuation of ammonium. In contrast, although the co-immobilized microalgae reactor showed the greatest ammonium
attenuation, it also showed the lowest nitrate production. This may be due to the fact that the symbiosis between bacteria and
microalgae may enhance the attenuation of the produced nitrates, probably through microalgal assimilation, as has been described in
wastewater, or directly by reducing nitrate formation (Casagli et al., 2021).

3.2. Batch experiment

Although reactors operated in continuous mode are more realistic than those operated in batch mode, batch studies can provide
valuable information regarding the impact of different reactor configurations such as the presence of microalgae or their co-
immobilization in MBBR carriers. Fig. 2 and Table 2 show that the removal efficiencies after 10 incubation days in the different
reactor configurations ranged from 74 % to 100 % for lincomycin and from 60 % to 98 % for ammonium. These efficiencies are similar
to those indicated above for the same reactors operated in continuous-feeding mode.
As occurred previously, the attenuation of lincomycin was complete; therefore, no differences were found in the total removal after
10 days of incubation between the reactors containing free or co-immobilized microalgae. Nevertheless, based on the kinetic rates (k)
and corresponding half-life times, differences were easily observed for lincomycin attenuation between free- and co-immobilized-
microalgae reactors (Table 2). The lincomycin kinetic rates for these two reactors were 0.32 and 0.85 d− 1, with half lives of
1.74–2.70 d and 0.64–1.02 d, respectively. These values are in the range of those observed for the attenuation of pesticides and an­
tibiotics in microalgae reactors fed with groundwater (Ferrando and Matamoros, 2020). In contrast, the control reactor with MBBR
carriers achieved a much lower k (0.18 d− 1), indicating that microalgae were indeed involved in lincomycin removal. Furthermore, the
removal efficiency of the co-immobilized microalgae was higher not only because those reactors contained higher amounts of biomass
than the other reactors but also because that biomass was the most efficient, as can be seen from the kbio values (Table 2). The kbio
values were higher for the co-immobilized microalgae, reinforcing the idea of a symbiosis between microalgae and bacteria on the

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Fig. 3. Peak intensities of lincomycin and its identified TPs over time in two types of reactors: 1) reactors containing diluted pig slurry (brown) and
2) reactors containing diluted pig slurry with co-immobilized microalgae on MBBR carriers (green).

biofilm created on the surface of the carriers.

Ammonium removal showed the same degradation trend as lincomycin. Degradation kinetic rates (k) were higher in the reactors
containing free or co-immobilized microalgae than in the control reactors (0.19 and 0.32 vs 0.12 d− 1, respectively), whereas the co-
immobilized reactors showed greater kinetic values than the free-microalgae reactors. Unlike lincomycin, the kbio of the different
reactors were similar but free microalgae achieved the highest kbio. This was also observed in the continuous operation experiment. In
any case, as ammonium removal was somewhat proportional to the amount of biomass, immobilization allowed to do so and thus
achieved higher ammonium removals.
According to the calculations made from the experimental data (see Section 2.3), 21 % of the overall kinetic removal observed in
the co-immobilized microalgae MBBR reactor was due to photodegradation and biodegradation in the diluted liquid phase of pig
slurry, whereas the presence of free microalgae increased that value by an additional 17 % and explained 38 % of the rate. Hence, 72%
of lincomycin was removed due to the microalgae co-immobilization.
Similarly, ammonium attenuation due to the processes occurring in the control reactors (nitrification due to bacteria) explained
38 % of the total kinetic rate observed in the co-immobilized-microalgae reactors, the presence of free microalgae explained 21 %, and
co-immobilization on MBBR carriers explained 41 % of the rate. This clearly shows that co-immobilization of microalgae could in­
crease the attenuation of ammonium and lincomycin compounds, probably by enhancing symbiotic interactions between microalgae
and bacteria. This is consistent with previous studies in which the immobilization of microalgae on different materials enhanced the
attenuation of antibiotics from groundwater (Ferrando and Matamoros, 2020), as well as of other microcontaminants from wastewater
(Solé and Matamoros, 2016).

3.3. Lincomycin transformation products (TPs)

Water samples from kinetic studies were injected into a UPLC-QToF. Two lincomycin TPs with an exact mass of 423.21555 were
identified at retention times (RTs) of 4.25 and 4.70 mins (Fig. 3). These compounds were the result of lincomycin oxidation on two
different sites, giving the same molecular formula at two different RTs but with different MS2 spectra (see Fig. S1, SM). The oxidation
product at an RT of 4.25 min had the same exact mass and MS2 fragments as a lincomycin TP previously reported by Zhou et al. (Zhou
et al., 2021). Also, the MS2 spectra of the oxidation product at RT 4.70 had one fragment matching the only MS2 fragment reported for
another lincomycin TP found by Zhou et al., although both spectra must be seen to ensure the match. Zhou et al. demonstrated that
both lincomycin oxidation TPs were the result of biodegradation and that they were produced by ammonium-oxidizing
microorganisms.
Zhou et al. also showed that lincomycin biotransformation in nitrifying environments resulted in several TPs (Zhou et al., 2021),
including the ones identified in the present study (oxidation products). This is consistent with the findings reported here, as the present
experiments were also conducted in a nitrifying environment (pig slurry). Furthermore, these compounds had a higher intensity in the
reactors with microalgae, where nitrification was faster, and their intensities increased with time (Fig. 3). This indicates, once again,
that part of the lincomycin was being oxidized together with ammonium (Fig. 2). Finally, as these two lincomycin TPs preserve their
structure, they could still maintain the microbial activity. The concentrations, toxicity and risks posed by these TPs should thus be
further evaluated.

3.4. Accumulation of lincomycin in biomass and potential revalorization

The concentration of lincomycin in the biomass of the reactors operated in continuous mode ranged from <LOD to up to
1500 ng g− 1 dw (dry weight was 2.5 % of the fresh weight). The concentration of lincomycin in the biomass of the control reactors
with MBBR carriers was lower than in the control reactors. Instead, for all the reactors with microalgae, lincomycin was below LOD
(Table 3). This can be explained by the described moderate lincomycin biodegradation and photodegradation in the environment

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M. Escolà Casas et al. Environmental Technology & Innovation 35 (2024) 103677

Table 3
Average and standard deviation of the concentration of lincomycin in the suspended solids or biomass contained in the different configurations
assessed in the continuous-operation mode (n=3). LOD of lincomycin was of 50 ng g− 1 dw.
Biomass (free) [g dw L− 1] Biomass (co-immobilized) [g dw L− 1] Lincomycin [ng g− 1
dw]

Control 0.40 ± 0.10 - 1073 ± 335

Control MBBR 0.51 ± 0.17 0.1 ± 0.07 754 ± 219
Free microalgae 0.72 ± 0.06 - <LOD
MBBR microalgae 0.55 ± 0.17 0.92 ± 0.50 <LOD

(Mehrtens et al., 2021). In contrast, although the biomass concentration was greater in the reactors with microalgae (0.72 g dw L− 1)
than in the control reactors (0.40 g dw L− 1), lincomycin was not detected in any of the samples containing algal biomass (Table 3). This
could be explained by the low uptake of lincomycin or by its high biodegradation due to the microalgae-bacteria consortium. Previous
studies working with microalgae have shown that accumulation of pharmaceuticals in microalgae is lower than in sewage sludge
(Matamoros et al., 2015). The current findings indicate that microalgae/biomass produced in the reactors contain significantly lower
lincomycin levels compared to biomass produced without microalgae.

4. Limitations of the study

The primary limitation of this study is the challenge of up-scaling. On the one hand, scaling photobioreactors is more complicated
than conventional wastewater reactors, as light intensity and availability are a critical factor (Benner et al., 2022). On the other hand,
diluting pig slurry for potential future expansion may necessitate recirculating outlet wastewater to dilute inlet pig slurry, introducing
logistical complexities and potentially increasing operational costs. Additionally, while nitrates in the water offer fertilization benefits,
careful management is crucial to mitigate potential environmental impacts. Also, further investigation into the formation of linco­
mycin or other antibiotic derivatives is required to assess transformation percentages and toxicity. Despite recent findings indicating
low levels of emerging contaminants in biomass from wastewater (Bellver et al., 2023; Álvarez-González et al., 2023), caution is
warranted regarding the potential presence of other pharmaceuticals.

5. Conclusions

This study demonstrates that co-immobilizing microalgae on MBBR carriers presents a viable solution for effectively removing
ammonium, nitrate, and lincomycin from the liquid fraction of pig slurry. Moreover, it results in the production of biomass with a low
concentration of lincomycin. Free-microalgae reactors operated in continuous mode at an HRT of 8 days increased the attenuation of
lincomycin (99 %) and ammonium (88 %) from pig slurry, whereas co-immobilization on MBBR carriers enhanced ammonium
removal to up to 94% and reduced the formation of nitrates from ammonium. Kinetic studies showed an increase in the removal rate
from 0.18 to 0.85 d− 1 for control and microalgae co-immobilized MBBR reactors, respectively. Reactors containing co-immobilized
microalgae produced the greatest amount of biomass. However, this biomass was in the form of biofilm, minimizing the increase of
suspended solids and COD in the outlet water. The accumulation of lincomycin in the suspended biomass of the control reactors ranged
from 750 to 1000 ng g DW− 1, whereas lincomycin was not detected in any of the microalgae reactors, whether free or co-immobilized.
In summary, this research highlights the efficacy of employing co-immobilized microalgae using MBBR carriers for the dual purpose of
lincomycin removal and high-value biomass production, without the presence of pollutants from pig slurry.

CRediT authorship contribution statement

Edward J. Pastor-López: Methodology, Data curation. Mònica Escolà Casas: Writing – review & editing, Writing – original draft,
Methodology, Investigation, Data curation. Víctor Matamoros: Writing – review & editing, Writing – original draft, Project admin­
istration, Investigation. Yolanda Rodríguez-Espelta: Methodology.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper

Data Availability

Data will be made available on request.

Acknowledgments

ME acknowledges the funding received through the Beatriu de Pinós 2018 grant-programme program (MSCA grant agreement
number 801370). IDAEA-CSIC is a Centre Severo Ochoa Center of Excellence Severo Ochoa (Spanish Ministry of Science and

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M. Escolà Casas et al. Environmental Technology & Innovation 35 (2024) 103677

Innovation, Project CEX2018–000794-S).

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.eti.2024.103677.

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