IMAGE SYSTEM FOR MICROALGAE CULTURE MONITORING IN A CUBESAT

Victor Ferreira​(1)​, Samuel P. Deccache Alves​(2)​, Gil R. V. Pinheiro​(3)​, Lia C.R.S.

Teixeira​(4)​, André L. S. Salomão​(5)

(1)
Dep. of Electronic and Telecommunication Engineering, Rio de Janeiro State
University, Brazil. ​victor.ferreira@ieee.org
(2)
Dep. of Electronic Engineering, Rio de Janeiro State University, Brazil.
samueldeccache123@gmail.com
(3)
Dep. of Electronic and Telecommunication Engineering, Rio de Janeiro State
University, Brazil. ​gilrpinheiro@gmail.com
(4)
Dep. of Sanitary and Environ. Engineering, Rio de Janeiro State University,
Brazil. ​lia.teixeira@uerj.br
(5)
Dep. of Sanitary and Environ. Engineering, Rio de Janeiro State University,
Brazil. ​andre@andresalomao.com

Keywords​: Chlorella vulgaris​, m

​ icroalgae cluster, image processing, remote
monitoring, CubeSat

Over the years, many studies have been carried out to ensure better conditions for
supporting human life on long-term missions in deep space or on planetary surfaces,
minimizing the need for resupplies. Microalgae are currently in space research,
mainly due to their high proliferation capacity, food production and even sewage
treatment. However, little is known about the real behavior (survival, biomass
production and sewage treatment capacity) of these microalgae under the effect of
microgravity and other space conditions. This project will aim to meet two
complementary research demands: to develop culture conditions for microalgae grow
in order to be monitore under migravity conditions and the development of an image
capture system for CubeSats, to monitor the microalgae cultures in real time. The
obtained results in a lab test, using the image acquisition and processing system
indicate the system ability to monitor the growing of the microalgae culture. As will be
presented, this system is apt to be installed in a CubeSat, to develop further studies
of microalgae under microgravity conditions. Microalgae cultures were monitore and
the colonies size were used as parameters for grow rates. The maximum production
of algae biomass for the three colonies evaluated were: 28% for C1 after 90 h of
monitoring; 21% for C2 after 84 h; and the greatest increase was seen in C3 with
36% after 120 h. ​The built system, for determining biological growth, composed of a
specific microscope and image processing algorithms, was able to determine the
evolution of the microalgae culture. A system arrangement was proposed, which can
be installed in a cubesat, for testing in a microgravity environment.Thus, the next step
is to test with microscope above the culture and an anti-fogging device and a better
illumination system.
1. Introduction

A CubeSat is a small satellite which performs a specific task when it is in orbit,

be it the observation of some phenomenon, where it can be physical, biological ​and
chemical, or, simply be a part of a data network around the Earth. In the design of
CubeSats, embarking on biological experiments is not a new subject [1], but it is
always a challenge, especially considering the difficulties in keeping organisms alive
and how to monitor them remotely.
A new perspective in the research area has been emerging, with a new concern
with the effect of untreated wastewater in spaceships that can cause serious
consequences in medium and long term missions. Whereas the new water and
wastewater treatment technologies are intensively developed to achieve the highest
organic and nutrient removal, wastewater treatment by phycoremediation process is
attracting interest because it is a low cost technology (operational and
infrastructure), because it has a high nutrient removal capacity and high rates of
biomass production [2]. Phycoremediation is defined as the use of algae to
biotransform substances, which may pose risks to the environment [3]. Some
unicellular green microalgae species, such as ​Chlorella vulgaris has been used in
studies of toxicity and wastewarter treatment in order to remove and biodegradate
drugs and other chemical compounds [2], [3]. ​Another advantage associated to the
use of microalgae in wastewater treatment is the ability of these microalgae to grow
together with other microorganisms, such as bacteria, making the treatment more
efficient in the removal and biotransformation of organic compounds and other
chemical compounds [4], [5]. Although extremophilles microorganisms, those able
to resist to adverse conditions in our planet (e.g extreme temperatures, high
pressures, radiation), are more suitable for astrobiology experiments, ​C. vulgaris ​is
a model organism for algae studies and perfectly fit to a preliminary studie for
methodologie development.
The culture medium of microorganisms must also be suitable for use in a space
environment, this will be one of the aspects addressed in the present work. For this
purpose, a system for determining biological growth was built, consisting of a
microscope for special purposes and image processing algorithms. Able to
determine the evolution of microalgae culture This work presents a proposal for the
remote monitoring of a microalgae culture in space, based on the analysis and
processing of digital images. To validate the solution presented, tests were
performed in the laboratory conditions in a solid medium and remotely monitored in
real time.

2. Materials and Methods

 2.1. Algae Culture

The unicellular microalgae, ​Chlorella vulgaris were cultivated and maintained in

incubators with photoperiod and temperature controlled according to the Standard
ABNT NBR 12648 [6] and the ISO 8692 [7]. The microalgae species cultivated in Rio
de Janeiro State University (UERJ) were purchased from the algae bank of the
University of Göttingen, Germany (SAG). Cultures were replicated at a monthly
frequency and growth and development of the cultures were monitored biweekly by
counting under an optical microscope [6]. Normally, microalgae cultures are
performed in a liquid medium, to allow their optical visualization in real time, it was
necessary to reduce the mobility of the cell colony. A solid medium with agar was
chosen.. A microalgae culture in solid medium view, under an optical microscope, is
shown in Figure 1.

Figure 1: A culture of microalgae ​Chlorella vulgaris in solid medium under an

optical microscope (400x magnification).

2.2. Biomass production

In this study, biomass production was evaluated by the remote sensing technique
in real time, after two different temperature conditions. Microalgae preculture was
stared five days before the tests, ensuring exponential growth in the inoculum
solution. Five petri plates (90 x 15 mm) containing 20 ml of oligotrophic medium
containing agar were inoculated with 10 µl of the inoculum solution of ​Chlorella
vulgaris​. The initial microalgae biomass applied for the test was 10​7​ cells.ml​-1​.
The test was divided in three steps of five days for the first and second steps and
seven days for the third step. The first step was performed with five petri plates and
under optimum conditions at 22 ± 2°C, with constant light (4500 lux). The second
step consisted of exposing all the petri plates at 4°C, at this temperature, the culture
reduced its metabolism, like in a long space journey. In the third step, the petri plate
with the highest growth of visual microalgae (i.e., biomass production) was chosen
and put under the optimum conditions of temperature and light (same conditions as
the first step), and the biomass production was evaluated, by the image acquisition
system in real time for seven days. The quantification of microalgae biomass was
performed by calculating the surface area of ​cell clusters, from the image of these
clusters in an optical microscope.

2.3. Image acquistion system for microalgae culture.

The system for determining biological growth was built, consisting of a

special-purpose microscope and image processing algorithms. The system was
based on previous work [8], where the author developed a similar system. The
system built was depicted in Figure 2, it was basically a digital microscope with
remote access. It comprised an optical system, consisting of a LED light source, a
10x microscope objective, a digital camera model Omnivision 5647, with resolution of
5MP and a stand with focus adjustment. The system was buit specifically for lab tests
with microalgae, but, aiming to be installed in ballons or Cubesats in future.

Figure 2: Picture of the microalgae culture and the image acquistion system.
 This remotelly operated digital microscope has been previously calibrated with a
special calibration scale and achieved a total magnification of 750 times and a field of
view (FOV) of 1 square milimeter. This system was used to test a microalgae culture
in the LABIFI lab, of UERJ. The imaging system acquired images of the culture
periodically, every 30 minutes, that were saved on a Single Board Computer (SBC),
running a Python program. The SBC can control the camera parameters and the light
source. The images were periodically sent to the monitoring computer (MC). The
SBC was a Raspberry PI 3 computer, and the MC was a notebook running MATLAB,
for image processing. As shown in Figure 2, the SBC had a video monitor, keyboard
and mouse, which could be used for laboratory tests.

2.4. Image processing

The microalgae cells were quantified using direct counting of image objects,
visually or through digital image processing algorithms [9]. In the present study, the
amount of cells was estimated by surface area. The Petri dish was 90 mm in
diameter and the microscope's FOV was limited to 1 square millimeter. The cell
growth rate was calculated by determining the area occupied by a group of cells.
Three cells colonies were chosen for this purpose (Figure 3).

Figure 3: Surface area of a microalgae colonies.

 The surface area of ​a microalgae colonie can indicate the amount of cells in a
limited way, as the cells can stack, growing in height instead of horizontally. In
addition to the occupied surface area, the number of cells contained in a specific
volume of solution is related to the area / volume ratio of the cells. Thus, smaller cells
have lower area / volume ratios. The calculation of the area of ​each cell colonie and
image processing were done using the MATLAB image processing toolbox and
specially developed scripts [8].

3. Results and Discussion

3.1. Monitoring issue and CubeSat overview

The SBC was operated remotely, through the MC, using a remote monitoring
software. As shown in Figure 4, the microalgae culture (red blots) was made over the
solid medium (green layer) inside the Petri Dish. The Petri dish had to be kept tightly
closed, to avoid contamination of the microalgae culture by fungi and bacteria. The
microalgae culture had a little moisture in the air space over the solid medium,
condensing internally on the upper surface of the Petri dish, like small drops of water,
making it difficult to see through the upper part of the petri dish. Thus, the microscope
objective was positioned under the Petri dish. Therefore, the images of the culture
were acquired through the thin layer of solid medium, as shown in Figure 3,
generating some loss in image quality.

Figure 4: Block diagram of image acquisition system, for lab tests with
microalgae ​C. vulgaris​.

In continuation of this research, to expose the microalgae culture to microgravity, a

microscope with remote operation will be prepared to be embedded in a balloon or
cubesat, as shown in the Figure 5. To strengthen the optical system, the light source,
a purpose built petri dish and the digital microscope will be mounted inside a tubular
stand. Which will be placed in a small culture oven, to control the temperature, and
avoiding to expose the microalgae to the extensive temperature variations, existing
outside CubeSat. A remotelly operated focus screw will position the microscope
inside the tube. Despiste the figure, the microscope can be mounted above the Petri
dish, if the internal temperature is maintained above the dew point, to avoid moisture
condensation inside the petri dish, and allowing the image acquisition by upper side.
A radio link with the ground station will support the image transfer and remote control
of the digital microscope, including the microalgae temperature, optical focus and
light intensity.

Figure 5: Block diagram of image acquisition system, for Cubesat tests with
microalgae.

3.2. Microalgae culture monitoring in real time

An area versus time graph was generated to assess the growth of microalgae, as
shown in Figure 6, the surface area is relative to the initial area (in the beginig of the
thrid step), when the test was started, at T = 0. The surface area of ​the cluster was
calculated every 6 hours. In order to know the average growth trend of the entire
microalgae culture, three groups of microalgae are compared (C1, C2 and C3) in the
same Petri dish and FOV (Figure 6).
 Figure 6: Surface area ​vs​ time of a microalgae colonies C1, C2 and C3.

In figure 6, there are five distinct phases in the microalgae colonies [9]: Latency
phase (Lag phase), relatively short phase, characterized by no growth or slow growth
(C1 and C3 T= 0 to 6 h), or even a decline in culture (C2 T= 6 to 18 h); Exponential
phase (Exp phase), there is a constant increase in the number of organisms in the
culture (C1 T = 6 to 90 h; C2 T = 18 to 78 h; C3 T = 6 to 108 h); Deceleration phase,
a decline in growth occurs, usually due to some limiting factor such as nutrients,
carbon dioxide or light (shading between cells) (C1 T = 90 to 102 h; C2 T = 78 to 96
h; C3 T = 108 to 114 h); Stationary phase, characterized by lack of growth and in a
short time the cells begin to undergo biochemical changes (C1 T = 102 to 120 h; C2
T = 96 to 132 h; C3 T = 108 to 120 h); and Decline or death phase, occurs when cell
metabolism can no longer be maintained due to depletion of nutrients or warning
conditions (C1 T > 126 h; C2 T > 138 h; C3 T > 126 h).
The maximum production of algae biomass verified with the increase in the area of
the solid medium for the three colonies evaluated was: 28% for C1 after 90 h of
monitoring; 21% for C2 after 84 h; and the greatest increase was seen in C3 with
36% after 120 h. It should be noted that the monitoring of the colonies occurred after
five days of storage of Petri dishes at temperatures of 4°C and in the absence of
light, few hours were necessay for the microalgae to break the dormancy and
showed satisfactory growth.
By comparing the images obtained over time with remote monitoring, it was
possible to observe that, after successive events of cell reproduction, the microalgae
located at the border of the clusters were smaller in size, when compared to the
microalgae in the centers of the colonies (Figure 1). This result was expected, since
the solid medium does not allow the dispersion of cells in the medium and, therefore,
the cells at the border of the clusters showed advantages in access to nutrients, gas
exchange and incidence of light (Figure 7) [10]. This more favorable condition
ensured a higher reproduction rate for these cells, resulting in a smaller size, due to
the reproductive processes of cell division. ​It is important to be aware that very high
microalgae densities (above 10​8 cell per mL) might cause self-shading and
accumulation of self-inhibitors [10].

Figure 7: Cut view of a microalgae cluster over the agargel.

4. Conclusion

Phycoremediation has become a promising technique, due to typical advantages of

biological treatments: low maintenance cost, absence of harmful chemicals and
environment friendly processes. Besides these advantages, phycoremediation also
produces biomass potentially useful for biofuel production.

The built system, for determining biological growth, composed of a specific

microscope and image processing algorithms, was able to determine the evolution
of the microalgae culture. A system arrangement was proposed, which can be
installed in a CubeSat, for testing in a microgravity environment.

Based on these preliminary results, additional experiments are proposed to test

different levels of relevant parameters, such as initial algal density, light intensity,
large scale of temperature and pressure and the efficiency of microalgae in
phycoremediation of wastewater after adverse environmental conditions. Thus, the
next step is to test with microscope above the culture and an ​anti-fogging device
and a better illumination system.

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