Algal Research 55 (2021) 102256

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

Microalgae classification based on machine learning techniques

P. Otálora a, J.L. Guzmán a, *, F.G. Acién b, M. Berenguel a, A. Reul c
a
Department of Informatics, University of Almería, CIESOL, ceiA3, 04120 Almería, Spain
b
Department of Chemical Engineering, University of Almería, CIESOL, 04120 Almería, Spain
c
Department of Ecology and Geology, University of Málaga, Campus de Teatinos, 29071 Málaga, Spain

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

Keywords: In this paper, two models for classification of microalgae species based on artificial neural networks have been
Artificial neural network developed and validated. The models work in combination with FlowCAM, a device capable of capturing each of
Deep learning the particles detected in a sample and obtaining a set of descriptive features for each one. One of the models uses
Image analysis
these feature variables as input, while the other makes use of the captured images, both being able to distinguish
Microalgae classification
FlowCAM
between two well-known species of microalgae, Scenedesmus almeriensis and Chlorella vulgaris, calculating the
proportion of each of these in the analyzed mixture. The models were trained with pure samples of each specie
and validated using mixed combinations of them. The results confirm the potential of image analysis and deep
learning techniques for the identification of microalgae cultures, as well as the higher accuracy of the feature-
based model, thus extending the range of classification approaches in this field.

1. Introduction
These days, society is facing several issues related to pollution and
the sustainability of the current lifestyle. The availability of clean water,
the emission of greenhouse gases and energy production are three
problems that will grow with population increase in the upcoming years.
In light of this scenario, the investigation of alternatives to the classic
solutions to these problems becomes mandatory, in order to find more
sustainable and efficient methods.
In this context, the production of microalgae at industrial scale ap­
pears to be a fairly interesting process. Microalgae are photosynthetic
microorganisms that can grow and reproduce in a wide variety of en­
vironments, without the need for fertile soil or clean water, being able to
grow even in polluted water [1]. The microalgae production is presented
as a suitable option for the abovementioned problem, since among its
applications lies the production of biofuels [2], as well as the fact that
the process itself is useful for wastewater treatment or CO2 emissions
mitigation from other industrial activities [3]. Furthermore, they have a
very beneficial composition for the production of high value products,
such as cosmetics, chemicals, human nutrition or animal feed [4,5].
Despite its multiple applications, it is a very complex process due to
its pronounced biological nature. Its productivity is determined by a
large number of variables related with the conditions of the culture and
the reactor design, and not all of them are controllable [4]. In order to
make the production process really competitive, it is essential to maxi­
mize productivity, and in order to do so, it is crucial to correctly char­
acterize the process [6].
A key aspect is the species of microalgae that is produced. Although
there are more than 30,000 known species of microalgae, less than a
hundred have been studied, and among these less than 20 are
commercially exploited [7]. Some examples are Chlorella, Spirulina,
Dunaliella or Nannochloropsis. Depending on the application for which
the process is intended, the use of one species or another, or even mixed
cultures, will be more advantageous. Therefore, a very important step in
optimizing the process is monitoring the culture composition [8].
Typically, this monitoring is performed by analyzing a sample with
an optical microscope. This is possible due to the morphological dif­
ferences of the different species of microalgae [9]. However, this re­
quires the participation of an expert capable of appreciating these
differences, while also being a complex and time-consuming activity
[10].
In previous works, different culture characterization methods based
on absorption spectroscopy or flow cytometry have been developed. The
methods based on absorption spectroscopy use spectrophotometers to
determine the pigment compositions of the samples, but they are
expensive [11]. Flow cytometry methods allow, in addition to

* Corresponding author.
E-mail addresses: p.otalora@ual.es (P. Otálora), joseluis.guzman@ual.es (J.L. Guzmán), facien@ual.es (F.G. Acién), beren@ual.es (M. Berenguel), areul@uma.es
(A. Reul).

https://doi.org/10.1016/j.algal.2021.102256
Received 23 October 2020; Received in revised form 20 January 2021; Accepted 20 February 2021
Available online 10 March 2021
2211-9264/© 2021 Elsevier B.V. All rights reserved.
P. Otálora et al.
determining the composition, cell count and the analysis of phyto­
plankton size structure [12]. Another approach is the optical image
analysis. This method allows individual classification of microalgae cells
based on their morphology [8]. However, it requires advanced image
capture and processing techniques.
In the last few years, machine learning techniques, and particularly
artificial neural networks (ANNs), have experienced a popularity in­
crease thanks to the growing computational capacity, and the large
volume of data that can be generated and managed nowadays [13].
These techniques have the ability to extract models based solely on data,
requiring less in-depth knowledge of the system and being able to adapt
properly to different circumstances. These models can make use of
different types of data, such as categories, numerical values or images,
depending on the problem to be solved. This type of techniques can be
also combined with particle analysis tools to obtain data that the ANN
will use as input.
Regarding the characterization of microalgae cultures, a neural
network model based on light absorption data was proposed in [14] to
distinguish between 4 species of microalgae. A convolutional neural
network in combination with FlowCAM is proposed in [15] to classify 19
different species. A hybrid method between image processing and ANN
is presented in [16] for identification and classification of 6 species. A
model based on deep learning techniques for detection and classification
of 5 different species is shown in [17]. The work in [18] proposed two
models to identify, classify and estimate the growth of microalgae using
support vector machine and random forest techniques. An image-based
neural network model for the classification of 16 different species was
developed in [19], capable of differentiating unique images of each cell
type with great precision. Several models with machine learning tech­
niques were proposed in [20] in order to differentiate living and dead
Chlorella vulgaris cells.
This paper develops two artificial neural networks to characterize
cultures composed by Chlorella vulgaris and Scenedesmus almeriensis,
which are one of the most commercial strains. The first of these ANNs
uses input data referring to the morphology of each one of the cells in the
sample, while the second makes use of the images of these cells, both of
them providing as output the composition of each one of the species in
the analyzed sample. All the data have been obtained with the tool
FlowCAM [21].
The main contributions of the work are a simple methodology that
can be easily generalized to other data acquisition devices. The fact of
having developed two ANNs provides great flexibility and adaptability
to the data to be used. The high volume of data used provides robustness
and reliability to the model, increasing its learning and verifying its
capacity of generalization. The validation of the model with mixed
samples is a very representative measure of the effectiveness of the
models developed. In addition, the application of detection thresholds
allows the classification of particles that do not correspond to micro­
algae cells.
The structure of the paper is the following. In Section 2, the tech­
niques used are presented, as well as the methodology that has been
followed. Section 3 describes the tests performed to obtain data, the
treatment of these data and the training process of the ANNs and their
validation. Finally, conclusions are given in Section 4.
2. Material and methods
This section presents the tools used to obtain and process the data, as
well as to develop the proposed models. As previously mentioned, the
FlowCAM tool will be used to obtain images and features of each one,
which will be used as inputs by the ANNs.
2.1. Microalgae species
The microalgae species that were used in the trials to develop the
network were Chlorella vulgaris and Scenedesmus almeriensis. Both are
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Algal Research 55 (2021) 102256
freshwater strains, characterized by its high growth rate and tolerance to
wide ranges of culture conditions such as pH, temperature and dissolved
oxygen. Thus, these strains are typical of open reactors including
coupling with wastewater treatment. Moreover, Chlorella vulgaris is one
of the microalgae strains more produced in worldwide for food related
applications, whereas Scenedesmus almeriensis has been reported as po­
tential source of lutein also for human related applications [22,23].
2.2. FlowCAM tool
FlowCAM is a device that analyzes particles or cells in a moving fluid
[21]. This tool takes photos and detects and counts the particles it de­
tects. Afterwards, it uses this information to analyze the particles to
extract a series of descriptive variables. The tool combines flow
cytometry, microscopy and fluorescence detection techniques. It is a
widely used tool in the field of biotechnology, making it a source of
easily acquired input data.
FlowCAM works as follows. A pump drives the sample into the fluid
chamber. In this chamber, the device constantly captures images of the
moving fluid with a period defined by the user. From these images, the
software detects, segments and processes each group of pixels repre­
senting a particle, thus obtaining the image of each cell and a series of
representative variables.
Each of these samples was first passed through a 20 μm mesh in
autoimage mode. The device was configured with a 20× lens, a cell size
of 50 μm and a flow chamber of 20 μm depth. A stop condition of 50,000
images is also imposed. Once set, the pump is activated and the image is
correctly focused. From here, the device automatically detects and
captures 50,000 images. For each of the samples mentioned, 3 tests are
performed as described, resulting in 150,000 images per sample.
FlowCAM provides as outputs images as those shown in Fig. 1 in .tif
format. As can be seen, it detects microalgae cells correctly, but has the
drawback that it can also detect undesirable particles, such as flocs or air
bubbles entering the pump. Another phenomenon that occurs and that
can be detrimental to the performance of the network is the agglomer­
ation of multiple cells in the same image. The data sheet provided by the
software already takes into account this phenomena in a column called
“ParticlesPerChain”, but the fact that these agglomerations can occur
between cells of different species, or even cells of one species and un­
desired bodies implies the need for using filtering techniques afterwards.
Besides these images, FlowCAM provides the features shown in Table 1,
as well as some others related to the date and time of image capture. It is
a tool capable of providing a large amount of information in a user-
friendly way, so it is appropriate for the problem faced.
2.3. Artificial neural networks
Artificial neural networks (ANN) are part of the group of algorithms
known as “machine learning”. These are characterized by being devel­
oped only from data, and are able to perform a given task without being
explicitly programmed for it [24]. Despite the fact that the computa­
tional cost of these algorithms is usually high, the growth of the com­
puter capacity in recent years has increased its popularity [13].
ANNs take their name from their similarity to the natural ones. They
are composed of a set of nodes divided into layers, where each node has
a series of inputs and outputs. The nodes of a given layer receive as input
the outputs of the nodes from the previous layer and provide as output a
function of these. This output is then used as input for the nodes of the
next layer, and so on until the last layer is reached [25].
A satisfactory ANN requires three fundamental parts to operate.
First, it is essential to have sufficient and adequate data for the training
of the network and its validation. The second fundamental component is
the structure of the network. The choice of the layers, the type and the
specific size are selected depending on the type of problem to be solved,
the type of inputs to be used, the number of data available, or the
complexity of the model to be developed. Finally, the last element to
P. Otálora et al.
Fig. 1. Sample images taken by FlowCAM of each species.
develop the model is the training process. This is defined by a series of
options such as its duration, the calculation frequency of the parameters,
the stop condition, but also which data will be used to train the network
and which will not [26].
In this work, ANNs will be developed for a classification problem,
comprising the abovementioned three parts as explained in Section 3.
The aim of this problem is that, given a series of entries, the ANN is able
to distinguish to which “class” these entries belong. Thus, for each series
of entries the network returns a value between 0 and 1 for each class.
The closer this value is to 1, the more likely it is that the classified entries
belong to that class. The sum of all outputs will always be equal to 1.
The ANNs and the data processing process of this work have been
developed using the “Deep Learning Toolbox” [26] and “Image Pro­
cessing Toolbox” [27] in the MATLAB environment, which is described
in the following section.
2.4. Deep learning and image processing toolbox
MATLAB’s “Deep Learning Toolbox” has been used for training and
validation of both ANNs. This toolbox allows the design of the ANN
structures layer by layer, establishing training options, carrying out that
training while providing relevant information about it, in addition to the
subsequent use of the trained ANNs for validation or prediction pur­
poses. It provides the user with an intuitive and powerful tool for the
creation of models based on deep learning techniques for different types
of problems [26].
The “Image Processing Toolbox” has a different functionality. It
provides the user with a large number of image processing, analysis and
display techniques, and can perform image segmentation, filtering or
batch processing tasks. This tool has been used to process all the data
used in the image-based ANN [27].
3. Results and discussion
This section presents the tests performed and the complete process to
obtain the ANN model. In addition, the results achieved in the training
and validation processes will be shown and discussed.
3.1. Performed tests
Data acquisition for ANN training and testing was performed by the
FlowCAM device. Two pure samples of Chlorella vulgaris and Scenedesmus
almeriensis were available. These samples were divided and mixed with
each other, in order to obtain different samples with different pro­
portions of each specie. Therefore, the samples analyzed by the tool
presented proportions from 1% to 99%, in addition to pure samples for
each species.
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Algal Research 55 (2021) 102256
The proportions were selected to represent very diverse samples,
with cases of balance between the two species, cases with one dominant
species over the other or extreme cases, such as 99/1, where the dif­
ference between concentrations is very significant. In this way it can be
checked whether the models developed are capable of correctly identi­
fying samples of all types.
In order to have a powerful and simple methodology, the idea is to
use only the pure samples for the ANN training process, since those are
the only ones in which it is known with absolute certainty to which
species of microalgae each particle corresponds. The rest of the combi­
nations have been used to test the ANN’s interpolation capabilities.
Notice that sometimes air is introduced into the pump, producing a
bubble that can alter the results. The FlowCAM software has a post-
processing tool that allows easily removing undesired images. These
images can be selected by size or by similarity with other images. From
this way, the most erratic images were removed, although this filtering
could have been done later. This process is relatively easy, but it requires
human intervention and is not possible to automatize it. In order to
minimize time consuming intervention only bubbles easily to detect
were removed. This intervention is justified by the fact that bubbles are
not part of the sample itself but is an artefact of data acquisition. In fact,
bubble retrieving should have been only necessary in the training
samples. This makes the final number of images in each sample slightly
less than 150,000.
3.2. Feature-based ANN
The first neural network to be developed will use as inputs the fea­
tures provided by FlowCAM from each of the detected particles (see
Table 1). As data acquisition was carried out in autoimage mode fluo­
rescence data were not available in this study. The acquisition of the
specific fluorescence per cell requires the presence of one cell in the flow
chamber when acquiring the image and data of each cell. If several cells
are in the flow chamber a mean fluorescence is associated to all cells.
Thus, fluorescence per cell values would require a very long acquisition
time. The autoimage mode in contrast allows the acquisition of all data,
except fluorescence, of 1–100 particles per frame. Therefore, in this
study autoimage and colour ratios were considered as proxy of cell
pigmentation.
3.2.1. Data processing
As mentioned previously, the ANN is able to learn directly from data.
For that reason, it becomes vitally important to guarantee the quality of
the available data. Using erratic or inappropriate data can lead to a
model that does not accurately represent reality. Therefore, before
training the network it is necessary to perform data processing.
For this ANN, the data processing process involved the selection of
P. Otálora et al. Algal Research 55 (2021) 102256

Table 1 known with certainty to which species each cell belongs. Using the data
Correlation index between each feature and the “Chlorella vulgaris” output [21]. sheets that FlowCAM provided after the tests, an input dataset was
Property Correlation Description created, consisting of 289,708 rows corresponding to each cell detected,
and 54 columns, corresponding to each of the properties of each cell. In
‘Area_ABD_’ − 0.5685 Number of pixels converted to a measure of
area addition, two more columns were added, one of which takes the value
‘AspectRatio’ 0.5054 Width/length 0 for the rows corresponding to Chlorella vulgaris samples and 1 for
‘AverageBlue’ 0.5587 Average pixel value of the blue color plane Scenedesmus almeriensis samples, while the other column always takes
‘AverageGreen’ 0.2451 Average pixel value of the green color plane the opposite value; 0 for Scenedesmus almeriensis and 1 for Chlorella
‘AverageRed’ 0.2837 Average pixel value of the red color plane
‘CalibrationFactor’ – Conversion factor from pixels to microns
vulgaris. Consequently, the initial input dataset have a size of 289708 ×
‘CalibrationImage’ – Number of the calibration image used in 56.
processing the particle However, the use of every cell property as an input is unnecessary
‘Camera’ – Camera number and may even have a negative effect on the performance of the ANN.
‘CaptureX’ 0.0311 Leftmost X coordinate of the particle in the
Thus, the correlation between each variable and one of the outputs will
original image
‘CaptureY’ − 0.0015 Top Y coordinate of the particle in the original be analyzed, in order to select the variables to be used. Table 1 presents
image the correlation between each of the inputs and the “Chlorella vulgaris”
‘Ch1Area’ – Area of fluorescence peak of photomultiplier output.
tube 1 As it can be seen, there are several properties that have no correlation
‘Ch1Peak’ Peak fluoresence value from photomultiplier
with the output. This is because they present constant values, so they do
–
tube 1
‘Ch1Width’ – Sample wifth of photomultiplier tube 1 above not provide information and thus are discarded. In addition to these, the
threshold value ‘CaptureX’, ‘CaptureY’, ‘ImageX’, ‘ImageY’ and ‘SourceImage’ proper­
‘Ch2Area’ – Area of fluorescence peak of photomultiplier ties were discarded, as they only provide information on the order in
tube 2
which the images were taken and this is not relevant information for the
‘Ch2Peak’ – Peak fluoresence value from photomultiplier
tube 2 network. The rest of the variables, regardless of their correlation, pro­
‘Ch2Width’ – Sample wifth of photomultiplier tube 2 above vide information about the cell morphology, so they will be considered
threshold value as inputs to the network. Fluorescence data were not available
‘Ch2_Ch1Ratio’ – Ch2Peak/Ch1Peak (explained above) therefore this data are not included. Since the ANN
‘CircleFit’ 0.3641 Deviation of the particle edge from a best-fit
will learn from data, it is not necessary to eliminate the variables with
circle
‘Compactness’ − 0.2988 Shape parameter derived from the perimeter lower correlation, since it will be the ANN itself that will give them less
and the area weight. Therefore, the final training dataset was reduced in size to
‘ConvexPerimeter’ − 0.6610 Approximation of the perimeter of the convex 289705 × 32 elements.
hull of a particle
Finally, in order to avoid problems associated with erratic sampling
‘Diameter_ABD_’ − 0.7189 Diameter based on a circle with an area equal
to the ABD area related to undesired particles, such as bubbles, an outliers filtering was
‘Diameter_ESD_’ − 0.6610 Mean value of 36 feret measurements applied to the dataset. This filtering eliminates the rows that present
‘EdgeGradient’ − 0.4227 Average intensity of the pixels of the outside significantly different values from the rest in any of the columns. This
border of the particle ensures that there are no erroneous data that alter the learning of the
‘Elongation’ − 0.2989 Lenght/breadth ratio based on perimeter and
ANN.
area
‘FeretAngleMax’ 0.0225 Angle of the largest feret measurement
‘FeretAngleMin’ 0.0970 Angle of the smallest feret measurement 3.2.2. ANN training
‘FilterScore’ – Statistical filter score Once the data has been processed, the structure of the ANN must be
‘ImageHeight’ − 0.5826 Image height in pixels in the collage
determined. As mentioned above, a classification problem is faced with
‘ImageWidth’ − 0.6271 Image width in pixels in the collage
‘ImageX’ − 0.1199 Leftmost X coordinate of the particle in the
two different classes: Chlorella vulgaris and Scenedesmus almeriensis. The
collage ANN will be trained only with data corresponding to pure samples of
‘ImageY’ − 0.2003 Top Y coordinate of the particle in the collage each species. Thus, it is known primarily that the ANN must have two
‘Intensity’ 0.3264 Average grayscale value of the pixels outputs. On the other hand, according to the variables analyzed above,
‘Length’ − 0.6960 Maximum value of 36 feret measurements
the input to the network must have a size of 30, corresponding to the
‘ParticlesPerChain’ 0.0223 Number of particles grouped into one
‘Perimeter’ − 0.5141 Total length of the edges of a particle number of features used. Therefore, the complete architecture of the
‘RatioBlue_Green’ 0.5998 Average blue/average green network will be composed of an input layer of size 30, a hidden layer and
‘RatioRed_Blue’ − 0.6579 Average red/average blue an output layer of size 2. After multiple trials with sizes between 10 and
‘RatioRed_Green’ 0.1339 Average red/average greeen
100 nodes, a size of 25 nodes was finally set for this layer. This layer size
‘Roughness’ − 0.3389 Measure of the irregularity of a particle’s
surface
has proven to perform very well while maintaining a simple and fast to
‘ScatterArea’ – Area of the peak of the scatter detector train structure. Fig. 2 represents the selected network architecture.
‘ScatterPeak’ – Peak value read from the scatter detector The next step in training is the division of the training dataset into
‘ScatterWidth’ – Sample width of scatter detector values above training, validation and test datasets. The training set will be the one
the threshold
from which the network will learn, and will therefore contain most of
‘SigmaIntensity’ − 0.3334 Standard deviation of grayscales values
‘SourceImage’ − 0.6209 Camera image number where the particle was the data. For the training set, 70% of the data will be selected, resulting
captured in a total of 202,796 samples. The purpose of the validation set is to
‘SumIntensity’ − 0.5562 Sum of grayscale pixel values check the network’s generalization capacity. The network will not learn
‘Transparency’ − 0.0366 1-(ABD diameter/ESD diameter) directly from this set, but it will be used to stop the training when the
‘Volume_ABD_’ − 0.1220 Sphere volume calculated from ABD diameter
‘Volume_ESD_’ − 0.0437 Sphere volume calculated from ESD diameter
generalization capacity does not improve. A 15% of the data (43,456
‘Width’ − 0.5710 Minimum value of 36 feret measurements samples) will be used for this purpose. Finally, the test set has no effect
on training, and it only provides a measure of the network’s perfor­
mance. The remaining 15% of the data will constitute this set.
input variables, the creation of output variables and the filtering of The network was trained using scales conjugate gradient back­
outliers. The first data to be processed has been the data for the training propagation. The training will last until the accuracy with the validation
process. These are the ones obtained from pure Chlorella vulgaris and set stops increasing for 6 iterations consecutively. This will mean that
Scenedesmus almeriensis samples, as they are the only ones where it is the ANN is memorizing instead of learning, which is detrimental to its

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P. Otálora et al. Algal Research 55 (2021) 102256

Fig. 2. Selected ANN architecture [26].

performance. During the training, 254 iterations were performed,
obtaining the results shown in the confusion chart in Fig. 3. This table
represents for each of the sets, as well as for the combination of all three,
the results of the prediction. The first two columns represent the actual
class to which an element belongs, while the rows show the class pre­
dicted by the network. Thus, the perfect prediction would be with 0 el­
ements in positions (2,1) and (1,2) of the table. Row 3 and column 3 are
summaries of the accuracy with which the ANN predicts each species
and its success rate with each species, respectively. In all sets, the pre­
diction error is approximately 3.1%, which can be considered highly
satisfactory with all the used sets.
3.2.3. Result validation
To completely validate the ANN it is necessary to check its accuracy
regarding the identification of mixed samples. For this purpose, the
datasets corresponding to the rest of the tests performed will be used.
The methodology will consist in introducing each row of the dataset as
input to the network, so that the ANN returns the output “Chlorella
vulgaris” and “Scenedesmus almeriensis” for each row. From these outputs,
which as previously mentioned can assume values between 0 and 1, it
will be detected how many of the rows correspond to each class, and
what is the overall proportion.
Therefore, the network was validated with each dataset corre­
sponding to the five different mixtures. The concentrations identified by
the network against the actual ones are shown in the first column of

Fig. 3. Confusion chart of the training process.

5
P. Otálora et al.
Table 2
Actual and predicted concentration for the ANN based on features with and
without threshold modification.
Actual Predicted concentration Predicted concentration
concentration without threshold with threshold
1%Ch–99%Sc 22.0–77.0 5.0–95.0
10%Ch–90%Sc 33.6–66.4 12.3–87.7
50%Ch–50%Sc 75.8–24.2 56.5–43.5
90%Ch–10%Sc 94.0–6.0 86.8–13.2
99%Ch–1%Sc 97.2–2.8 93.5–6.5
Table 2. As can be seen, the predicted concentrations are significantly
different from the actual ones. For almost all the mixtures, the network
predicts a much higher percentage of Chlorella vulgaris than the actual
one. Considering that the prediction error in pure samples was around
3%, it can be deduced that the error when measuring the concentration
does not lie in the accuracy of the ANN when classifying a cell.
Thus, there can be other sources of error. As mentioned above,
FlowCAM does not only detect microalgae cells, but also any particle
larger than the configured cell size. This implies that there are rows
corresponding to undesired particles. Furthermore, the grouping of
several cells that FlowCAM can detect in a single row, either of different
species or of the same species, is also a factor that can result in a worse
identification. Fig. 4 shows a histogram of both outputs for the 50/50
sample dataset. As can be seen, the network detects a very high number
of cells with a Chlorella vulgaris output very close to 1, while with Sce­
nedesmus almeriensis it is more uncertain. This can be due to the fact that
FlowCAM detects Chlorella vulgaris cells more easily, or that the ANN has
a tendency to detect Chlorella vulgaris when different types of particles
join together.
Therefore, it is necessary to perform a tuning that achieves a balance.
So far, when counting the cells of each type, a cell has been considered to
be Chlorella vulgaris as long as its Chlorella vulgaris output is greater than
its Scenedesmus almeriensis output, i.e. greater than 0.5. Nevertheless,
since the ANN tends to detect more Chlorella vulgaris than there really is,
the threshold above which a cell is considered to be Chlorella vulgaris was
chosen incrementally. This was changed to 0.5 to 0.995. This value was
selected in view of the histogram shown in Fig. 4, and by trial and error
steps. The implementation of the threshold also allows the detection of
particles that do not belong to any of the species, being those for which
the output does not exceed any of the two thresholds.
Following this adjustment, the concentrations determined by the
network are those shown in the third column of Table 2. As can be seen,
Fig. 4. Histogram of the ANN output for the 50/50 sample.
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the results are very close to the actual concentrations, as well as being
symmetrical, which certifies that the threshold has been correctly
imposed. Fig. 5a and b shows the real concentration of each species
compared to that determined by the ANN, for each of the 3 tests per­
formed with each concentration. The ANN behaves in a very similar way
for each test on the same sample, forming practically a unitary slope
line.
3.3. Image-based ANN
The second neural network developed use as inputs the images
captured by FlowCAM from each of the detected particles, providing a
more general classification method.
3.3.1. Data processing
In this case, data processing is equally or even more important than
for the ANN developed earlier. As already mentioned, this network will
work from the .tif files provided by FlowCAM, reason why the steps that
will be followed for its development will be the segmentation of images
and their resizing to ensure a homogeneous input size.
Since there is no way to separate the individual images directly from
the FlowCAM, it is necessary to divide them afterwards. For this purpose,
MATLAB’s Image Processing Toolbox has been used with a script that can
accomplish this objective. The script transforms the image to grayscale,
and by imposing a threshold of intensity, filters each pixel with a value
of intensity higher than that threshold. Once only these pixels are ob­
tained, the different images are separated, detecting as independent
images those whose pixels are not in contact with each other. Then, once
each individual image has been detected, its position in the initial image
is obtained and it is cut out of it, saving it as a separate image in .png
format. In this way, each image is available to be treated as an individual
input by the network. This process was performed with the images
corresponding to all the samples, automating and simplifying image
treatment. As can be seen in Fig. 1, each image has a different size, while
the network that has been used needs uniform size inputs, all the images
were resized to 227 × 227 pixels.
3.3.2. ANN training
As done for the other ANN, the next step is the training stage. Given
that this network works with a different type of data than the previous
one, it is essential to determine a different architecture. Since it is an
image classification network, it was decided to start from the structure
of an existing network. That is AlexNet, a convolutional ANN composed
of 25 layers shown in Table 3. For this specific case, the size of the input
layer is 227x227x3, color images are used, and the size of the output
layer is 2. This ANN belongs to the group of algorithms known as deep
learning, due to its depth and the complexity of the used layers.
The dataset available for the training, which is composed by the
images corresponding to the unmixed Chlorella vulgaris and Scenedesmus
almeriensis samples, will be divided using 80% of the images for the
training set and the remaining 20% for validation. In this case, no test set
will be used, Since images are more complex data and it is convenient to
employ more resources in training. In addition, it is a long process, so the
division into three sets would only make the training longer. Certain
training options will also be established such as the maximum training
duration, which will be 3, the initial learning ratio, which will be 0.001,
and the validation patience, also used in the training of the other
network, which will adopt a value of 5 iterations.
This training process is significantly slower than the previous one,
exceeding 4 h in duration, because the size of the entries is much larger,
as well as the number of layers in the network. After the training process,
the final error of the trained network is 1.6% with the validation data.
This is a reasonably low error, and so it is possible to claim that the
network is able to accurately predict which class each image belongs to.
P. Otálora et al. Algal Research 55 (2021) 102256

Fig. 5. Predicted concentration against actual concentration for both species and both ANNs.

mixed samples. The methodology in this case is the same as the previous
Table 3
one. The input dataset of each sample is provided and the images of each
Layers of the neural network for image classification [26].
class classified by the network are counted, obtaining the composition of
Number Layer type the mixture. The results obtained at first instance are shown in Table 4.
1 Image input Similar to the previous case, these are not particularly accurate, as the
2 Convolution network has a tendency to predict a higher percentage of Chlorella vul­
3 ReLU garis than the actual one. The justification for the error remains the same
4 Cross channel normalization
5 Max pooling
as before: undesired particles or particle clusters, or even a tendency for
6 Grouped convolution FlowCAM to detect Chlorella vulgaris cells more frequently.
7 ReLU In view of this circumstance, the previous solution was considered. A
8 Cross channel normalization detection threshold was applied, so that an image will only be consid­
9 Max pooling
ered as Chlorella vulgaris when its similarity to this species exceeds this
10 Convolution
11 ReLU threshold. After several iterations, the threshold with the best results
12 Grouped convolution was determined to be 0.9999975. To obtain this value, the default
13 ReLU threshold of 0.5 was used as a starting point, gradually increasing this
14 Grouped convolution value. In each iteration, the predicted ratio for the 50/50 set was
15 ReLU
16 Max pooling
checked until it was found a value that provided satisfactory results.
17 Fully connected From here, this value was tested with the other sets, completing its
18 ReLU validation. This means that for the network to decide that an image is
19 Dropout
20 Fully connected
21 ReLU Table 4
22 Dropout Actual and predicted concentration for the neural network based on images with
23 Fully connected and without threshold modification.
24 Softmax
Actual Predicted concentration Predicted concentration
25 Classification output
concentration without threshold with threshold

1%Ch–99%Sc 25.5–74.9 1.2–98.8

3.3.3. Result validation 10%Ch–90%Sc 34.6–65.4 10.9–89.1
Following the network training, as in the previous case, it is imper­ 50%Ch–50%Sc 77.0–23.0 56.4–43.6
90%Ch–10%Sc 95.4–4.6 89.7–10.3
ative to test its performance when identifying the compositions of the 99%Ch–1%Sc 98.6–1.4 97.5–2.5

7
P. Otálora et al.
Chlorella vulgaris, it must be completely certain of this. Consequently, the
identification of both species becomes balanced and in the same way as
in the previous case, the detection of particles not belonging to any of
the species or aggregates of cells is achieved at the same time.
Through this methodology, the results summarized in the third col­
umn of Table 4 are obtained. The ANN has the capacity to represent
reality with considerable precision, with a minimum difference between
the true and the predicted composition. Fig. 5c and d shows the graphs
representing the predicted concentration against the real one is shown
for each of the tests performed with each sample. The dispersion be­
tween the predictions is quite small, and the line relating both axes is
very similar to the unitary slope, which would be the perfect prediction.
3.4. Discussion
After the development and validation of both ANNs, both have
proven to be capable of overcoming the proposed task. The identifica­
tion of microalgae cultures formed by two different species has been
successfully accomplished using two different types of data, which val­
idates the usefulness of ANNs for this kind of problems. A great volume
of data has been used for both the training and the validation steps.
These data belong to three different trials for each sample, and in all
cases the results have been very precise. The classification of images
proves to be fairly fast, with a classification time of less than 0.001 s per
sample for the feature-based network, and 0.1 per sample for the image-
based network, respectively (processor: Intel(R) Core/TM i7-9700 CPU
@ 3.00 GHz; RAM: 16.0 GB).
When comparing the two networks with each other, both have fol­
lowed a similar methodology but have shown slightly different results.
The ANN that has used images as input has achieved more accurate
results, which is at first sight the most important aspect. However, this
network also requires much more time to be trained and to classify any
sample, since it is more dense and uses more complex data. The images
are also heavier data, being more complicated to be shared. Therefore,
the use of one network or another is justified by the compromise be­
tween precision and complexity. Another factor that can be taken into
account is the ease of obtaining data, because although in this work
FlowCAM is able to provide both, if it is desired to use another data
acquisition tool, this fact could change and the ANN using images as
inputs allows generalization to other devices.
4. Conclusions
This paper presents two ANNs for identification of microalgae sam­
ples composed by Chlorella vulgaris and Scenedesmus almeriensis. The
networks use as input data images of microalgae cells and descriptive
features provided by the tool FlowCAM. In both networks, training has
been done with pure samples and a classification threshold tuning has
been applied. The training error did not exceed 4%, while in validation
the maximum error was 6.5%. Regarding future work, it is interesting to
extend the network by using a greater number of species and samples in
different conditions, as the followed methodology makes it easy to
generalize the classification problem to more species.
Statement of informed consent, human/animal rights
“No conflicts, informed consent, human or animal rights applicable”.
Declaration of authors
All the participants are authorship of this work and agree to submit
the manuscript for peer review to Algal Research.
CRediT authorship contribution statement
Otálora P. was the responsible of the work, the neural network
8
Algal Research 55 (2021) 102256
development, and the writing of the manuscript. Guzmán J. L. was the
responsible of the model adaptation and the parameter tuning. Acién F.
G. contributed to the elaboration of the manuscript and revision.
Berenguel M. was the responsible of the Discussion section. Reul A.
contributed to revision and finalization of the manuscript.
Declaration of competing interest
The authors declare any potential financial or other interests that
could be perceived to influence the outcomes of the research.
Acknowledgments
This work has been partially funded by the following projects:
DPI2017 84259-C2-1-R (financed by the Spanish Ministry of Science and
Innovation and EU-ERDF funds), and the European Union’s Horizon
2020 Research and Innovation Program under Grant Agreement No.
727874 SABANA.
The acquisition of the FlowCAM by the University of Málaga was co-
financed by the 2008–2011 FEDER program for Scientific-Technique
Infrastructure (UNMA08-1E005).
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