Algal Research 50 (2020) 101983

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Year-long assessment of a pilot-scale thin-layer reactor for microalgae T

wastewater treatment. Variation in the microalgae-bacteria consortium and
the impact of environmental conditions
⁎
A. Sánchez Zuranoa, , J.A. Garrido Cárdenasb, C. Gómez Serranoa, M. Morales Amarala,
F.G. Acién-Fernándeza, J.M. Fernández Sevillaa, E. Molina Grimaa
a
Chemical Engineering Department, University of Almeria, Ctra. Sacramento, s/n, 04120 Almería, Spain
b
Biology and Geology Department, University of Almeria, Cra Sacramento s/n, 04120 Almería, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: This work studies microalgae production using urban primary wastewater as the only nutrient source in a pilot-
Microalgae scale thin-layer cascade photobioreactor (TLPBR). The experiments were carried out by operating the photo-
Bacteria bioreactor in semi-continuous mode at a constant dilution rate (D = 0.3 days−1) uninterrupted over ten months.
Wastewater The TLPBR was effective at removing the main nutrients (nitrogen, phosphorus and chemical oxygen demand,
Thin-layer
COD) with biomass productivities ranging from 28.3 g·m−2·day−1 to 47.3 g·m−2·day−1 over the different sea-
Metagenomics
sons of the year. In addition, the evolution of the cells' photosynthetic efficiency (photosystem II) was also
Nutrient removal
analysed. The removal of the main nutrients was correlated with the changes in the microbial-bacteria con-
sortium existing in the culture system. These microbial fluctuations were correlated with the temperature and
the solar irradiance impinging on the culture surface. The most abundant bacterial phyla were Proteobacteria
and Bacteroidetes with the most dominant order being Rhodobacterales and Sphingomonadales, belonging to
the Alphaproteobacteria class. Functional analysis of the relationship between bacterial populations, removal of
major nutrients and the environmental parameters showed that the three main bacteria orders together (41% of
the total) represent the main bacteria groups, which remained over the months tested; however, their abundance
was affected by fluctuations, which might be explained by environmental variations and major nutrients such as
the chemical oxygen demand (COD).

1. Introduction microalgal-bacterial interactions in the phycosphere, the microscale

Conventional technologies for wastewater treatment have restric-
tions concerning their high-energy requirements, poor nutrient removal
performance and environmental impact due to greenhouse gas emis-
sions. In this regard, environmentally friendly and low-cost alternatives
to the convection-activated sludge processes have appeared over the
last two decades, especially those using microalgae processes [1–4].
The microalgae strains used in wastewater treatment are characterised
by their rapid growth and tolerance to irradiance and temperature.
Strains such as Oscillatoria, Scenedesmus, Chlorella and Nitzchia have
been established as the most pollution-tolerant microalgae in waste-
water treatment systems [5]. Applying microalgae to wastewater
treatment involves them associating with other aerobic and anaerobic
microorganisms. These associations have been demonstrated in natural
environments; for instance, various researchers have described the
area surrounding microalgae cells where metabolites are exchanged
between microalgae and bacteria [6,7]. Regarding microalgae waste-
water processes, the microalgae-bacteria interactions were first de-
scribed by Oswald et al. [8]. Nowadays, a variety of interactions be-
tween algae and bacteria have been described (mutualism,
commensalism, parasitism, etc.), which can range from beneficial to
detrimental to algal growth [9]. According to the mutualistic relation-
ships, they may improve nutrient removal in wastewater processes.
Different groups of bacteria synthesize important compounds for algal
growth, including micronutrients, siderophores, growth stimulants and
antibiotics to protect against possible pathogen invasions [10]. On the
other hand, aerobic bacteria remove oxygen from the microalgae's
microenvironment, which might be toxic to the microalgae cells. In
addition to consuming dissolved oxygen, bacterial respiration produces
carbon dioxide, which can be immediately used for microalgal

⁎
Corresponding author.
E-mail address: anasanchezzurano@gmail.com (A. Sánchez Zurano).

https://doi.org/10.1016/j.algal.2020.101983
Received 26 March 2020; Received in revised form 22 June 2020; Accepted 23 June 2020
2211-9264/ © 2020 Elsevier B.V. All rights reserved.
A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

photosynthesis [11,12]. Despite the benefits from these microalgae- Table 1

bacteria associations, bacteria that negatively affect algal growth also Average composition of the culture medium and primary domestic wastewater
appear in the microalgae wastewater reactors. These bacteria influence used as the influent in the thin-layer reactor. Concentrations expressed as
microalgal activity through the synthesis of secondary metabolites, mg·L−1. COD: chemical oxygen demand, TC: total carbon; TN: total nitrogen;
TP: total phosphorous.
which are strong algicidal agents [10]. Even though there are methods
that remove associated bacteria, it is not possible to maintain bacteria- Parameters Primary domestic wastewatera Arnon media
free algal cultures in large-scale culture systems, especially in outdoor
pH 7.9 ± 0.3 7.5 ± 0.2
open ponds [11]. It is clear, therefore, that the microorganisms in the
COD 511.0 ± 100.9 16.0 ± 1.2
microalgae-bacteria consortium could have unpredictable and some- Sulphate 98.1 ± 6.4 6.3 ± 0.8
times detrimental effects on microalgae growth and the efficient de- Nitrogen-Nitrate 4.0 ± 2.7 140.0 ± 4.5
toxification of wastewater pollutants, which are essential considera- Chloride 411.6 ± 23.5 78.9 ± 2.1
Sodium 222.5 ± 12.1 276.1 ± 7.9
tions for sustainable microalgae wastewater treatment [13,14]. Despite
Potassium 8.6 ± 1.6 325.1 ± 6.3
microalgae population performance being a function of culture condi- Calcium 30.1 ± 0.2 364.9 ± 5.5
tions, fluctuations in wastewater composition, and biotic and abiotic Magnesium 54.1 ± 14.1 12.2 ± 0.6
parameters [3], scarce information is available regarding bacterial po- Phosphorus-Phosphate 12.5 ± 5.1 39.3 ± 3.1
pulations. Moreover, it is thought that the abundance of most groups in Nitrogen-Ammonium 75.9 ± 17.7 0.0 ± 0.1
Iron 0.19 ± 0.01 5.0 ± 0.3
these systems is determined by the wastewater used [15].
Copper 0.09 ± 0.07 0.02 ± 0.0
Currently, there are several demonstration facilities, such as the Manganese 0.03 ± 0.01 0.5 ± 0.02
Christchurch Wastewater Treatment Plant (New Zealand) [16] or the Zinc 0.10 ± 0.08 0.06 ± 0.01
Chiclana Wastewater Treatment Plant (Spain) (http://www.all-gas.eu/ Boron 0.35 ± 0.09 0.4 ± 0.03
en/), investigating microalgae-bacteria wastewater treatment using TC 288.0 ± 13.2 52.4 ± 4.9
TN 79.9 ± 18.1 140.0 ± 4.5
open culture systems, such as raceway reactors, with culture depths TP 12.5 ± 5.1 39.3 ± 3.1
ranging from 10 to 30 cm. However, thin-layer cascade technology,
which was established in the 1970s, has proven to be generally more Values from the Primary domestic wastewater correspond to the mean ± SD
productive in daylight than raceway reactors, at least at the small scale (n = 10) and values from the Arnon media correspond to the mean ± SD
[17]; nonetheless, this has not been comprehensively evaluated for (n = 3).
a
microalgae-bacteria wastewater treatment in terms of biomass pro- This is the average composition of wastewater used during the experiments
ductivity, removal capacity and biological diversity. period.
In view of this, some authors have suggested that it is necessary to
improve the knowledge of the bacterial communities involved in mi- 2.2. Photobioreactor and operating conditions
croalgae processes (wastewater treatment being one of them) using
various molecular biology techniques to monitor these populations in An open 32 m2 thin-layer cascade reactor operated at a 0.02 m
real-time and then try to regulate them [11,18]. Metagenomic analyses water depth was used (Fig. 1). The reactor was equipped with a 0.8 m3
are particularly useful techniques for better understanding the com- sump where pH is controlled at 8 by the on-demand injection of pure
plexity and dynamics of microalgae-bacteria interactions [13,19]. This CO2 at 5 l min−1, or air supplied at 50 l min−1, to remove oxygen. In the
work attempts to assess (a) the use of thin-layer cascade technology in thin-layer reactor, the total culture volume was 1.6 m3. The culture was
microalgae wastewater treatment, and (b) the bacteria-community di- circulated at 0.2 m s−1 using a pump that pushes the culture up to the
versity and structure appearing in a thin-layer reactor when treating first layer, lifting it 0.5 m from the culture level in the sump [20].
wastewater coming from primary treatment (PTWW), and how this A SCADA system monitors and controls the reactor's overall op-
changes over the seasons of the year. In order to reach these goals, the eration including environmental parameters such as solar radiation and
removal of the major nutrients contained in the wastewater inflow was ambient temperature, and culture parameters, such as pH (#5333,
evaluated. These data allowed to correlate any changes in the microbial Crison, Spain), temperature (#PT1000, Crison, Spain) and dissolved
community profile to nutrient consumption. In addition, these potential oxygen (#9336, Crison 9336, Barcelona, Spain). The experiments were
microbial fluctuations were correlated with the environmental para- performed in semicontinuous operational mode in the TLPBR. For this,
meters measured, such as temperature and radiation. Illumina MiSeq the reactor was inoculated with a 10% total volume of Scenedesmus sp.
platform was used for the high-throughput sequencing of the 16S rRNA culture. Then, the reactor was filled up with wastewater from primary
gene to analyse and characterise the entire microbiome of the micro- treatment (PTWW) and operated in batch mode for a week. Following
algae wastewater samples. this, the reactor was operated in semicontinuous mode at 0.3 day−1
(i.e., every morning 30% of the reactor volume was harvested over 3 h,
approximately, while an equal volume of PTWW was introduced over
2. Materials and methods the same period; the monthly volume of PTWW used was about 10 m3).
This semicontinuous operational mode was maintained throughout the
2.1. Microorganisms and culture conditions ten months of trials. The use of PTWW involved varying its composition
over the months of the experiment. At certain times when changing the
The freshwater Scenedesmus sp. strain was used. Scenedesmus has PTWW tank, adjustments had to be made and the batch operational
been widely reported for outdoor microalgae production because of its mode was reinstated for a week until re-establishing the conditions that
tolerance to adverse conditions. For the experiments, the inoculum was allowed the reactor to operate in semicontinuous mode (at the 0.3 day
−1
prepared using modified Arnon medium as the standard, with the cul- dilution rate). Samples were taken each week for steady-state
ture medium prepared using fertilizers instead of pure chemicals. The characterization and metagenomic analysis. The experiments were
inoculum was transferred to a pilot-scale outdoor TLPBR by initially performed from March to December in Almería (36°48′N, 2°43′W),
filling the reactor with primary wastewater. The average composition of Spain.
the culture medium and the wastewater used is reported in Table 1. The
primary wastewater was obtained from an urban wastewater treatment 2.3. Biomass concentration, fluorescence measurement and analytical
plant in Almeria (Spain), operated by FCC AQUALIA S.A. methods

The biomass dry weight concentration (Cb) was measured daily by

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 A. Sánchez Zurano, et al.

3
Fig. 1. The thin-layer system (32 m2) operate at a 0.02 m of culture thickness. The reactor is equipped with an aerated sump from which the microalgae culture is led through to the first layer using a centrifugal pump. A)
Scheme of the thin-layer cascade reactor located at the Cajamar Experimental Station “Las Palmerillas” along with the mounting dimensions. B) Thin-layer cascade reactor 3D model.
Algal Research 50 (2020) 101983
A. Sánchez Zurano, et al.
filtering 100 ml of culture through filtered through Macherey-Nagel
glass fiber MN 85/90 and drying it in an oven at 80 °C over a 24 h
period. Biomass productivity (Pb) was calculated multiplying the bio-
mass concentration by the dilution rate. In addition, the maximum
photosynthetic efficiency of photo-system II, calculated as the Fv/fm
ratio, was measured using a fluorometer (AquaPen AP 100, Photon
Systems Instruments, Drasov, The Czech Republic) to check the phy-
siological status of the cells.
The standard official methods approved by the Spanish Ministry of
Agriculture were used to analyse the water composition in the reactor
[21]. Phosphorus was measured by visible spectrophotometry through
the phospho–vanado–molybdate complex. Nitrates were quantified
spectrophotometrically at 220 and 275 nm. Ammonium was measured
using the Nessler method. Sodium, calcium, potassium, magnesium,
iron, copper, manganese and zinc were determined by atomic absorp-
tion. Chloride was measured using a volumetric method with silver
nitrate between 0.01 and 1 N; potassium chromate was used as the
indicator (The Mohr Valuation). Boron was determined spectro-
photometrically following a reaction with azometine. Sulphates were
measured using a precipitate of baric sulphate. Chemical oxygen de-
mand (COD) was determined by spectrophotometric measurement
using HachLange kits (LCK-555).
2.4. DNA extraction and 16S rRNA gene amplicon sequencing library
preparation
In order to assess the microbial community's evolution over the ten
months in the thin-layer reactor, the bacterial genomic DNA was ex-
tracted from all the collected samples and used as the template.
Metagenomic analyses were performed using the Illumina MiSeq
system. The V3 and V4 variable regions of the 16S rRNA gene were
amplified from the bacterial DNA obtained from the wastewater bio-
mass samples. Then, PCR reactions were performed using specific pri-
mers. Subsequently, the size of the products from the PCR was eval-
uated using an Agilent 2200 TapeStation (Agilent Technologies, Italy).
The PCR products were purified by discarding primers and primer di-
mers using the Agencourt AMPure XP Kit (Beckman Coulter Genomics,
Italy). Dual indices and Illumina sequencing adapters (P5 and P7) were
then attached to the amplicons using the Nextera XT Index Kit
(Illumina, San Diego, CA) to develop the final libraries using the
Illumina 16S Metagenomic Sequencing Library preparation protocol.
The PCR reaction conditions were as follows: 95 °C for 3 min,
8 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a final
extension step at 72 °C for 5 min. Subsequently, the final libraries were
quantified as absolute using KAPA Library Quantification Kits for
Illumina® platforms (Kapa Biosystems Ltd., UK). The diluted library
sample concentrations were then calculated against the standard curve
using the absolute quantification. The final libraries were pooled in
equimolar amounts, denatured and diluted before being loaded onto
the MiSeq flow cell and sequenced on the Illumina MiSeq platform
(Illumina, San Diego, CA) with MiSeq reagent Kit v3 (Illumina, San
Diego, CA) [22].
2.5. Bioinformatics analysis
Finally, the raw sequencing data were processed using the QIIME
pipeline [23] using the “closed-reference” OTU picking strategy. Raw-
reads quality was checked using FastQC v0.11.2. The quality control
was performed using QIIME, and the reads were collected into OTUs
(Operational Taxonomic Units (with an identity ≥97%) using the
QIIME closed-reference OTU picking strategy against the QIIME-for-
matted Greengenes v.13.8 reference database (http://greengenes.lbl.
gov). Finally, the sequences were clustered into the 3 main taxa ranks
(phylum, class and order).
The Qiime alpha-diversity programme was used to calculate Alpha
diversity. For the calculation, the normalization was performed using
4
Algal Research 50 (2020) 101983
the “rarefaction” QIIME process. The alpha diversity was determined
using the ‘observed species’, ‘Chao1’ (the estimator of species richness),
the ‘Shannon diversity index’ and the ‘Simpson Diversity Index’.
2.6. Data analysis
Statistical analysis was performed using the Statgraphics Centurion
software programme. To compare the influence of nutrient removal and
environmental parameters (temperature and radiation) values on the
statistically significant microbial community structures, multivariate
analysis was performed with a 95% CI for the means of each variable. P
values less than 0.05 were considered statistically significant.
3. Results and discussion
3.1. Biomass productivity and nutrient removal efficiencies
Different steady states were obtained over the ten months of the
experiments along with variations in the system performance evaluated
with regard to biomass productivity, light utilization efficiency and
nutrient removal capacity, as well as the metagenomics analysis. The
composition of the PTWW varied over the experimental period; hence,
the average composition of all the PTWW used was calculated and
evaluated. The PTWW contained relevant amounts of minor elements
such as Fe, Mg, Mn, etc. - all necessary for microalgae production
(Table 1). Regarding the major compounds, the PTWW contained high
chemical oxygen demand (COD) levels, above 511 mg·L-1. Regarding
the nitrogen and phosphorous concentrations, the PTWW contained
nitrogen that was mainly in the form of ammonium (75.9 mgN-NH4·L-1)
and 12.5 mgP-PO2− 4 ·L-1. Analysing these concentrations, a N/P ratio of
6.15 was observed. This ratio was compared with the biomass chemical
composition that had a relative C/N/P composition of 100/11/2. This is
a N/P ratio of 5.5 in the biomass. The comparison between the biomass
and PTWW N/P ratio shows that the use of this medium might result in
phosphorous limitation with respect to the nitrogen content, which in
turn might affect the elemental composition of the biomass. However,
this phenomenon can change according to the specific composition of
the biomass produced [20]. It was assumed that no phosphorous pre-
cipitation took place since the culture's pH values and the low P-PO4−3
ion concentration made such precipitation unlikely [24].
In order to compare the seasonal biomass productivity and to ana-
lyse any possible changes in the bacterial populations, data on im-
pinging solar radiation (Fig. 2A) and temperature (Fig. 2B) were re-
gistered over the months of the experiment. With regard to areal
productivity (Fig. 2C), it was distinguished between the heterotrophic
biomass productivity and the photoautotrophic part. For the hetero-
trophic productivity calculations, the typical stoichiometric parameters
for the biomass yield on COD in aerobic activated sludge from ASM
(activated sludge model) were taken and applied [25]. The rest of the
biomass productivity was considered photoautotrophic biomass. Data
showed that the heterotrophic part is a very small proportion and that
the photoautotrophic productivity is dominant, ranging from
26.6 g·m−2·day−1 to 45.9 g·m−2·day−1. Maximal productivity was ob-
tained in May, June and July, corresponding to the highest daily ra-
diation values reached in Almeria. For the rest of the months, the areal
productivity ranged from 28.3 g·m−2·day−1 to 33.9 g·m−2·day−1; this
supported high production system stability. Despite the potential
phosphorous limitation imposed when PTWW was used as the nutrient
source, the results showed that it was possible to achieve a high level of
biomass productivity from March up until the end of the year, and to
ensure its stability. The productivity obtained during the ten months of
the TLPBR experiment was significantly higher than that for raceway
reactors using wastewater (10–20 g·m−2·day−1) [26]. The productiv-
ities described were greater than those recorded by the third-generation
thin-layer cascades (660 m2 in area) located in Třeboň (Czech Republic)
for the period between May and September, in which the average
A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

Fig. 2. Annual variation in radiation, temperature, biomass productivity and fluorescence of chlorophylls in outdoor semicontinuous cultures of Scenedesmus sp.
cultivated in a thin-layer reactor. A) Average of solar radiation per day each month. B) Temperature. C) Areal biomass productivity. D) Variation in fluorescence of
chlorophylls as a function of the time of the year. Values correspond to the mean ± SD (n = 3).

biomass productivity observed ranged from approximately
14 g·m−2·day−1 to about 17–18 g·m−2·day−1 [17]. These data suggest
the importance of weather conditions in microalgae productivity. In
Almería, the high productivities are the results of the favourable
weather conditions throughout the year.
The stability of the biological system was reflected in the Fv/fm
values recorded. The physiological status of the microalgae cells was
checked daily during the experiment and the steady-state data for each
month are displayed in Fig. 2D. The results showed that the Fv/fm
remained almost constant during the test. However, the Fv/fm

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A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

Fig. 3. Influence of the month of the year on the culture medium showing the inlet and outlet nitrogen and phosphorous concentrations, along with the nitrogen and
phosphorous removal, in outdoor semicontinuous Scenedesmus sp. cultures cultivated in a thin-layer reactor. A) N-NH4+ at the inlet and outlet. B) N-NO3− at the
inlet and outlet. C) P-PO4 at the inlet and outlet. Values correspond to the mean ± SD (n = 3).

parameter decreased to below 0.6 in May, June and July, probably due
to the high radiation values measured in Almeria. Photoinhibition in
microalgae cultures at high irradiances has been reported both in out-
door and indoor cultures [27], and this has caused reversible damage of
key PSII components [28,29]. Over the rest of the time, the Fv/fm rose
to the 0.6–0.7 range, which illustrates that Scenedesmus sp. has con-
siderable tolerance to temperature and radiation oscillations.
Regarding nutrient removal, the reactors' inlet and outlet flows were
analysed, and mass balances were applied. The results showed a wide
−1
variation in the N-NH+4 inflow (50.4–106 mg·L ). Despite this varia-
+
bility, the N-NH4 was consumed entirely by the culture, with the
outflow being between 0.2 and 7.6 mg·L−1 N-NH+ 4 . After applying the
balance, it was observed that the culture's N-NH+
4 consumption ranged
from 15 to 30.6 mg·L−1·day−1 (Fig. 3.). These consumption values
correspond to a depuration efficiency greater than 90%. Despite the
high efficiency of the system, one must consider that specific

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A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

competition could appear between the microalgae and the ammonia- photosynthetic efficiency of photosystem II (Fv/fm) was at its lowest.
oxidizing bacteria in metabolizing the ammonium from the PTWW However, for the rest of the months, a direct correlation was observed
[30]. Furthermore, this idea is supported because the nitrate con- between the inflow and the consumption, demonstrating the consortia's
sumption is lower than the ammonium consumption. The N-NO3− in capacity for removing organic matter.
the inlet was determined and evaluated, showing lower entry values in To summarise, looking at the results for the productivities, the Fv/
the TLPBR, between 0.7 and 9.1 mg·L−1. This relates to the N-NO3− fm and the nutrient removal from the thin layer reactor, it is evident
consumption, which ranged from 0 to 2.2 mg·L−1·day−1. Despite the that the microalgae-bacteria system demonstrated robust stability over
low input concentrations, the N-NO3− was still visible at the outlet the ten consecutive months, with the high removal capacity and op-
(0.9–3.3 mg·L−1), probably as it was not consumed entirely or because timal physiological status being especially influenced by environmental
it was generated by nitrifying bacteria (Fig. 3. B) [31]. Given the results changes.
concerning the low nitrogen concentrations at the outlet, it was possible
to confirm that the proposed microalgae wastewater treatment com-
3.2. Microbiota community diversity and structure
plied with the effluent discharge limits (European Directive 91/271/
CEE).
In addition to evaluating the effectiveness of the thin-layer cascade
Phosphorus plays a key role in the metabolism of both microalgae
technology in terms of biomass productivity and nutrient removal for
and bacteria because it can be incorporated into organic molecules such
use in microalgae wastewater treatment, useful metagenomic tools
as DNA, RNA or lipids through phosphorylation [32]. Phosphorous
were employed to determine the bacterial diversity present in the thin-
removal presented a similar pattern to that observed for N-NH+ 4 (Fig. 3.
layer reactor. The fastq sequence files from Illumina MiSeq were ana-
Thus, when the P-PO4−3 in the inlet increased, the consumption in-
lysed using QIIME software. After filtering for quality, trimming length,
creased as well. The P-PO4−3 concentration in the wastewater feeding
and assigning taxonomies, the phylogenetic variation was studied over
the TLPBR showed high variability (6.6–20.1 mg·L−1), with consump-
the test period. All the samples were successfully categorised into their
tion inside the reactor being virtually total, leading to P-PO4−3 fluc-
phylum, class and order. Considering only the taxa that had a relative
tuations in the outflow of between 0 and 3.8 mg·L−1. Hence, by ap-
abundance of more than 1%, the microbial community overall was
plying a biomass balance, the phosphorous consumption varied from
mainly comprised of 13 phyla, 19 classes and 31 orders.
1.8 to 5.6 mg·L−1·day−1 over the evaluation period. The highest con-
Following rarefaction, which normalised to the sample with the
sumption values were found during the months when the P-PO4−3 in
lowest number of sequences, the species number observed per sample
the wastewater entering the TLPBR increased, confirming that there
was between 1594 and 2382. In order to understand the bacterial
was potential phosphorous limitation.
structure of the samples, both diversity and richness were measured;
The chemical oxygen demand (COD) was also analysed at the re-
this was done by developing a variety of bioinformatics tools. Richness
actor inlet and in the harvested culture throughout the ten months of
is defined as the number of different microorganism types present in a
the trial. It should be pointed out that the COD forecast in the reactor
niche, and it can be measured with Chao1, a non-parametric method
inlet was from 383.5 to 654.5 mg·L−1 (Fig. 4). Similarly, the COD in the
that is useful when species with low abundance appear in a niche [33].
outlet was evaluated, where it ranged between 41.6 and 220 mg·L−1.
Diversity, on the other hand, not only considers the microbial richness
From these data, it is possible to observe that the COD did not directly
of a niche, but also gives us information on evenness, which, in turn,
increase in the outlet when the COD percentage at the inlet increased;
compares the relative importance of each of the species present. Two of
this could indicate that the TLPBR was capable of depurating high COD
the most used measurements are the Shannon and Simpson diversity
concentrations. The maximum COD value at the inlet was 654.5 mg·L−1
indices due to their robustness. Both values provide additional in-
in August with the corresponding COD value at the outlet being 112.5,
formation to the simple richness or uniformity of the species in a niche,
meaning the consumption rate was 162.6 mg·L−1·day−1. These data
with each index providing different information. While the Shannon
show that it was possible to attain an 83% depuration efficiency when
index focuses on species richness, the Simpson index focuses instead on
there were high COD values at the inlet. During the other months, COD
species uniformity. The Simpson index can give values between 0 and 1,
consumption fluctuated between 81 and 178.3 mg·L−1·day−1. There-
the value being greater the smaller the diversity, whereas the Shannon
fore, the COD results show that strong consumption was possible every
index can present any value, and is greater the more species there are in
month in spite of the high inlet values that sometimes appeared, except
a medium; also, its distribution is more uniform [34].
in June, when the consumption was approximately half that of the inlet;
The number of observed species as well as the species richness index
this coincided precisely with the month of the trial when the
(Chao1) was slightly affected by the time of year, probably due to the

Fig. 4. Influence of the time of the year on the culture medium for the inlet and outlet chemical oxygen demand, along with the chemical oxygen demand removal, of
outdoor semicontinuous Scenedesmus sp. cultures cultivated in a thin-layer reactor. Values correspond to the mean ± SD (n = 3).

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A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

Table 2 orders determined. The results were in accordance with previous au-
Number of reads per sample assigned to OTUs, and the alpha diversity metrics thors who have suggested that bacteria within Rhodobacterales present
of the treated wastewater's microbial community over the months of the ex- a clear trend that can be associated with phytoplankton in general, and
periments. to increases in phytoplankton blooms [19,43]. More specifically, spe-
Month Numbers of reads Chao1 Observed species Shannon Simpsons cies of the Roseobacter genus, belonging to Rhodobacterales, are het-
erotrophic bacteria that are commonly observed in close association
March 625,758 2978 2382 7.18 0.94
with phytoplankton because are consumers of organic carbon during
April 625,200 2801 2180 6.68 0.93
May 624,632 2624 1978 6.22 0.93
and after algal blooms [44]. Moreover, they have been described for
June 597,966 2591 1989 6.36 0.92 producing signalling compounds that preventing bacterial infection
July 814,115 2325 1754 6.24 0.93 during prosperous algal blooms [45]. The second most abundant class
August 592,130 2131 1594 5.93 0.91 were Betaproteobacteria, including Burkholderiales, which had a high
September 578,574 2288 1691 5.92 0.92
variability (3–16%). The presence of Burkholderiales in the microalgae
October 565,018 2445 1789 5.90 0.93
November 366,172 2491 1887 6.50 0.94 wastewater bioreactor might be justified given that several bacterial
December 468,290 2643 2003 6.00 0.88 strains from the Burkholderia genus are N2-fixing bacteria and promote
growth both in plants and in microalgae [46]. These changes in pro-
portions were also seen in Xanthomonadales, which belong to the
weather conditions (Table 2). Concerning Shannon and Simpson in- Gammaproteobacteria; these were more prevalent in September and
dexes, no significant differences between the samples were observed. October. Concerning the Bacteroidetes phylum, orders such as Flavo-
These data indicated an important bacterial diversity in these com- bacteriales, Cytophagales and Saprospirales were detected in all the
munities. samples. Despite it not being possible to generalize and say that the
Fig. 5A shows the phylogenetic classification of the phylum and bacterial composition of the microalgae phycosphere is species-specific,
class discovered in the experiment. Proteobacteria were the most sig- most bacterial populations are often restricted to just a few bacterial
nificant phylum, ranging from 55% to 70% over the months of the test. groups, including members of the Alphaproteobacteria, Gammapro-
Bacteroidetes were the second most abundant phylum in the samples, teobacteria and Flavobacteriia [47,48]. The results showed two other
fluctuating between 8% and 20%. Indeed, Proteobacteria and Bacter- relevant orders within the Bacteroidetes phylum: Saprospirales and
oidetes have been reported to be the most predominant phyla in various Cytophagales, although at low percentages. These two groups are
activated sludge processes [35,36]. In addition, these are the major among the four major groups of algicidal bacteria - Cytophaga, Sa-
bacteria in marine environments [37]. Although the exact role of prospira, Pseudoalteromonas and Alteromonas. They are capable of
members from Proteobacteria and Bacteroidetes phyla in the bacteria/ degrading cell walls, killing the microalgae by breaking down the cell
microalgae consortium is unknown, it is postulated that it is funda- surface macromolecules. Previous authors have suggested that the de-
mental. It is probably associated, in its natural state, with the de- tection of Cytophagales and Saprospirales in bioreactor samples might
gradation of organic matter, dissolved and in particles, derived from mean that prolonged contact between the microalgae introduced into
marine phytoplankton [38]. Furthermore, previous phylogenetic assays the wastewater and the endogenous microflora could induce predatory
have demonstrated that bacteria belonging to the Proteobacteria and bacteria [14]. However, the results obtained in this work showed that
Bacteroidetes phyla are more likely to establish interactions with green Saprospirales abundance decreased over the months, while the maximal
algae (such as Scenedesmus sp.) than are other bacterial phyla [39]. abundance of Cytophagales (10%) in November corresponded with an
Firmicutes were the next most commonly occurring phylum detected in elevated microalgae cell status (Fv/fm = 0.67) and biomass pro-
all the samples (3–6%) over the test period, and this predominance is ductivity (33 g·m−2·day−1), suggesting that the microalgae population
evidence of the availability of volatile fatty acids, such as butyrate and was not affected by these algicidal bacteria.
its analogues. Since Firmicutes are considered to be fermenters of these The presence of other important minority orders, such as
simple volatile fatty acids, the wastewater used must be a source of Rhizobiales (alphaproteobacteria), Pseudomonadales (gammaproteo-
these biodegradation products [35]. Actinobacteria were the next most bacteria) and Chromatiales (gammaproteobacteria), which are im-
abundant phylum detected in all the samples, ranging from 2% to 5%. plicated in many biological processes, was especially low. The presence
They play an important role in wastewater treatment processes because of Rhizobiales has been justified by previous authors [19] who in-
they perform denitrification activities along with filamentous fungi dicated that Rhizobiales are common contaminants of green algal cul-
[40]. Various other minority phylum classifications, such as Plancto- tures, contributing 50% of the bacterial load in axenic Chlamydomonas
mycetes and Chloroflexi, were present at small percentages. Whereas reinhardtii, Chlorella vulgaris and Scenedesmus sp. cultures. Moreover,
the percentage of Plantomycetes remained practically constant bacteria belonging to Rhizobiales could be algal growth promoters.
throughout the year, the percentage of Chloroflexi increased from Au- However, this positive effect is not appreciated in the Pseudomonadales
gust to October. There were also other minority phylum classifications order, which has been described as exerting alga-lytic activity towards
present at small percentages such as Verrucomicrobia, Acidobacteria, microalgal biomass in wastewater lagoons [49]. Although the presence
Gemmatimonadetes, OD1, Spirochaetes, SR1 and TM7. Previous au- of Chromatiales was low during the experiment, they are an important
thors have reported that these less predominant species might play an bacteria group in microalgae processes because they include ammonia-
essential role in stabilizing wastewater treatment processes [41,42]. oxidizing bacteria such as Nitrosococcus [50]. The maximal abundance
The bacterial class classification (Fig. 5B) was dominated by Pro- of Chromatiales (4%) was observed in July, corresponding to high
teobacteria from the alpha, beta, gamma, delta and epsilon subclasses, concentrations of N-Nitrate in the outflow (3.27 mg·L-1). These data
with the alpha, beta and gamma classes being the most abundant might indicate that nitrification occurred that month.
subdivisions of Proteobacteria in the samples. The percentage of bac-
teria belonging to the Flavobacteria varied over the course of the ex- 3.3. In-depth analysis of the evolution of the microbiota community
periment (2–14%). In addition, another Bacteroidete, Saprospirae was structure and the relationship with nutrient removal and environmental
significantly more present at the start of the study. However, Clostridia, parameters
which belong to Firmicutes, remained steady over time (2–3%).
To take a closer look at the evolution of the microbial community In order to explain the variability in the microbiota structure during
structure, the metagenomic data was further analysed and compared at the test, a statistical analysis was carried out using the nutrient con-
the order level (Fig. 6). Rhodobacterales and Sphingomonadales, be- sumption values and the environmental parameters (Table 3). In par-
longing to the Alphaproteobacteria class, were the most abundant ticular, it was evaluated the influence of nutrient consumption

8
A. Sánchez Zurano, et al.
Fig. 5. Seasonal variation in taxonomic classification of the bacterial communities at the phylum level (A) and at the class level (B).
(ammonium, nitrate, chemical oxygen demand and phosphorous) and
the environmental variables (temperature and radiation) on the main
order levels detected by metagenomics analysis.
Regarding the Rhodobacterales order, its distribution over the test
period was determined by the radiation received (p = .04). As men-
tioned already, Rhodobacterales is a highly diverse group, which in-
cludes bacteria with a wide range of metabolisms (aerobic respiration,
anaerobic fermentation, sulphur oxidation, autotrophic carbon fixation,
nitrogen fixation or hydrogen production). Within this order, two of the
most relevant groups are the bacteria that perform anoxygenic photo-
synthesis, and the photoheterotrophic bacteria, which depend on
9
Algal Research 50 (2020) 101983
oxygen to photosynthesise [51]. Their distribution over the months was
strongly influenced by the radiation received (p = 0,04). This re-
lationship was supported by [52], who reported that the activity of
phototrophic groups such as Rhodobacterales, increased along with
UVR, suggesting that photo-activated repair mechanisms are present
that can resist high levels of solar radiation. However, no relationship
has been established with nutrient removal. In contrast, another re-
levant order, Sphingomonadales, showed a significant correlation be-
tween radiation (p = .04) and COD consumption (p = .01). Sphingo-
monadales belong to the Gammaproteobacteria class, which is known
for including a group of photoheterotrophic species. This bacteria group
A. Sánchez Zurano, et al. Algal Research 50 (2020) 101983

Fig. 6. Seasonal variation in taxonomic classification of the bacterial communities at the order level.

Table 3
Multivariate analysis was performed with 95% CI for the means of each variable. P values of less than 0.05 were considered statistically significant.
Order Overall (%) (> 1) p-value

Removal (mg/L) Environmental Parameters

Phosphorus COD Ammonium Nitrate Radiation Temperature

Rhodobacterales 17 > 0.05 > 0.05 > 0.05 > 0.05 0.04 > 0.05
Sphingomonadales 14 > 0.05 0.01 > 0.05 > 0.05 0.04 > 0.05
Burkholderiales 10 > 0.05 < 0.01 > 0.05 > 0.05 > 0.05 > 0.05
Xanthomonadales 9 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Flavobacteriales 6 > 0.05 0.04 > 0.05 > 0.05 > 0.05 > 0.05
Cytophagales 4 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Saprospirales 3 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Rhodocyclales 3 > 0.05 > 0.05 > 0.05 > 0.05 < 0.01 0.02
Clostridiales 3 > 0.05 > 0.05 > 0.05 < 0.01 > 0.05 > 0.05
Acidimicrobiales 3 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Pseudomonadales 2 0.03 > 0.05 > 0.05 > 0.05 > 0.05 0.02
Rhizobiales 2 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 0.01
Bacteroidales 2 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Rhodospirillales 2 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Chromatiales 2 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Campylobacterales 1 0.02 > 0.05 0.02 > 0.05 > 0.05 > 0.05
Verrucomicrobiales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 0.03
Actinomycetales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 < 0.01
Pirellulales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Caldilineales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Solibacterales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Chloroflexales 1 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Spirochaetes 1 > 0.05 > 0.05 > 0.05 0.03 > 0.05 < 0.01

10
A. Sánchez Zurano, et al.
present a predominantly chemoheterotrophic metabolism, but this is
supplemented with light energy using photosynthetic reaction centres
composed of bacteriochlorophyll. A previous study looking at these
organisms concluded that in alpine lakes, where harsh conditions pre-
dominated (such as high levels of UV radiation, low temperatures, and
low concentrations of dissolved organic carbon (DOC) and nutrients),
the bacteria detected were almost exclusively Sphingomonadales spe-
cies [53,54]. These results concur with the results proposed in this
work, suggesting a direct correlation with radiation and an indirect
correlation with COD consumption. As with the previous order, mem-
bers of the Sphingomonadales order were noted many years ago as
exhibiting minimal DNA damage and a high level of UV-B resistance
[55].
Another abundant Proteobacteria, Burkholderiales, showed a sig-
nificant relationship (p < .01) to the COD consumption. The principal
member of the Burkholderiales order is the Burkholderia genus, which
can be characterised by its functional diversity and versatility. The most
known species from this group are studied because of their potential in
promoting plant growth; this is because they can convert atmospheric
nitrogen to ammonia via biological nitrogen fixation. There are other
important members used for bioremediation since they are catabolically
versatile, thus, enabling them to degrade recalcitrant xenobiotic com-
pounds, and to survive in environments with limited nutrient avail-
ability [56,57]. In this last context, Burkholderia is considered one of
the dominant genera for performing organic pollutant degradation in
wastewater [58]. The strong correlation between Burkholderiales and
COD consumption in the data is probably due to their capacity for
eliminating organic compounds in waste. Considering the abundance of
these three main orders together (41%), it is possible to explain why the
main bacteria groups were maintained during the test months; none-
theless, their abundance is affected by both environmental parameters
and nutrient concentrations. No significant correlation was found with
the Xanthomonadales order. Data showed that order level Flavo-
bacteriales, belonging to the Bacteroidetes phylum, were influenced by
phosphorous consumption (p = .04), with the highest proportion of
Flavobacteriales found at high phosphorous removal values. However,
for Cytophagales and Saprospirales, it was found that neither this re-
lationship, nor any other, were present in the parameters studied. The
most abundant order, Clostridiales, belonging to the Firmicutes
phylum, showed a significant relationship with the monthly nitrate
consumption (p < .01) – there was an inverse correlation between the
Clostridia percentage and nitrate removal. This decrease could be ex-
plained by the nitrate in the medium because nitrate salts significantly
suppress sporulation in members of the Clostridiales order [59]. Re-
garding the order levels described that were present in lower propor-
tions, it is important to emphasise the significant correlation that was
determined between Rhizobiales (p = .01) and Pseudomonales
(p = .02) with temperature variation. The proportions of these orders
were lower during the warmer months. The latter, Pseudomonales,
showed another relationship with the phosphorous consumption
(p = .03). This relationship is due to the presence of phosphatase en-
zymes, which are responsible for much of the recycling of organic
phosphorus in bacteria belonging to the Pseudomonadales order [60].
Moreover, temperature showed a significant correlation with other
orders present in low proportions, such as Rhodocyclades, Verrucomi-
crobia, Actinomycetales, Sphingobacteriales and Spirochaetes. These
results are comparable with previous authors who found that deni-
trifiers in wastewater, such as Rhodocyclales, and Pseudomonadales,
were favoured when the temperature was lowered from 35 °C to 25 °C
[61].
The results obtained in this study suggest that environmental
parameters influenced both the fluctuations in the main bacteria po-
pulations detected and the microalgal activity, which was reflected in
the productivity and Fv/Fv results. Nonetheless, the nutrients in the
inflow and their consumption were important for many of the bacteria
groups; even though their influence on the microalgae would be in
11
Algal Research 50 (2020) 101983
question given that the system was highly effective despite the varia-
tions in nutrient concentrations. Therefore, this study revealed that
microalgae-bacteria systems used for wastewater treatment made up a
robust consortium, the stability of which may be due to maintaining the
same dilution rate over the ten months of the trial. Furthermore, the
consortium was capable of withstanding extreme fluctuations in the
PTWW composition and the environmental variables.
4. Conclusions
This study presented initial research on the seasonal evolution of the
biomass productivity, nutrient removal and associated bacterial com-
munities for the valorisation of urban wastewater treated with micro-
algae in a 32 m2 thin-layer cascade reactor, along with identifying the
parameters influence them the most. The study represents a powerful
initial work for improving knowledge regarding microalgae-bacteria
association and for modelling its activity. In order to develop and op-
timize microalgae-bacteria processes using this technology, the results
suggested that microalgae-bacteria systems demonstrated great stabi-
lity despite variations in the PTWW composition and changes in the
environmental conditions. Over the ten months of the trial, the main
bacteria groups were maintained and the productivities in the reactor
were high. Future work should involve the scaling-up of this technology
to a reactor surface that is hundreds of m2. In this regard, should a
different aspect ratio be used for the cascade channels of the scaled
TLPBR other than that presented in Fig. 1, there may be changes in the
channel culture depth, flow rate and eventual gradients in temperature,
pH, and dissolved oxygen in the different zones of the reactor that
should also be evaluated.
CRediT authorship contribution statement
A. Sánchez Zurano:Conceptualization, Data curation, Writing -
original draft.J.A. Garrido Cárdenas:Validation, Writing - review &
editing.C. Gómez Serrano:Investigation, Formal analysis.M.
Morales Amaral:Investigation, Data curation.F.G. Acién-
Fernández:Conceptualization, Validation, Funding acquisition.J.M.
Fernández Sevilla:Software, Supervision, Funding acquisition.E.
Molina Grima:Writing - review & editing, Funding acquisition.
Declaration of competing interest
There are no potential financial or other interests that could be
perceived as influencing the outcome of the research. No conflicts, in-
formed consent, human or animal rights issues are applicable. All the
authors confirmed authorship of the manuscript and agreed to submit it
for peer review.
Acknowledgements
This research was funded by the SABANA project (grant # 727874)
of the European Union's Horizon 2020 Research and Innovation
Programme; the PURASOL project (CTQ2017-84006-C3-3-R, Ministry
of Economy and Industry, Govt. of Spain); the Marine Microalgal
Biotechnology group (BIO 173) in the Chemical Engineering
Department (University of Almeria, Spain); Fundación Cajamar and the
Spanish Ministry of Education through the National FPU Programme
(grant number FPU16/05996). Furthermore, this work was supported
by the Bioinformatics Core Facility at PTP SCIENCE PARK (Lodi, Italy).
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