Control of predators in industrial scale microalgae cultures with Pulsed
Electric Fields

D. Rego, L.M. Redondo, V. Geraldes, L. Costa, J. Navalho, M.T. Pereira

PII: S1567-5394(14)00120-0
DOI: doi: 10.1016/j.bioelechem.2014.08.004
Reference: BIOJEC 6774

To appear in: Bioelectrochemistry

Received date: 17 January 2014

Revised date: 28 July 2014
Accepted date: 12 August 2014

Please cite this article as: D. Rego, L.M. Redondo, V. Geraldes, L. Costa, J. Navalho,
M.T. Pereira, Control of predators in industrial scale microalgae cultures with Pulsed
Electric Fields, Bioelectrochemistry (2014), doi: 10.1016/j.bioelechem.2014.08.004

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Control of predators in industrial scale microalgae cultures with

Pulsed Electric Fields
D. Regoa, L. M. Redondob,*, V. Geraldesa, L. Costaa, J. Navalhoa and M. T. Pereirac
a
A4F - Algafuel S.A., Lisbon, Portugal

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Lisbon Engineering Superior Institute, ISEL, Lisbon
c

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EnergyPulse Systems, EPS, Lisbon, Portugal

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Abstract

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This work describes the utilization of Pulsed Electric Fields to control the protozoan contamination of
a microalgae culture, in an industrial 2.7 m3 microalgae photobioreactor. The contaminated culture
was treated with Pulsed Electric Fields, PEF, for 6 hours with an average 900 V/cm, 65 s pulses of 50

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Hz. Working with recirculation, all the culture was uniformly exposed to the PEF throughout the
assay. The development of the microalgae and protozoan populations was followed and the results
showed that PEF is effective on the selective elimination of protozoa from microalgae cultures,
inflicting on the protozoa growth halt, death or cell rupture, without affecting microalgae
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productivity. Specifically, the results show a reduction of the active protozoan population of 87%
after 6 hours treatment and 100% after few days of normal cultivation regime. At the same time,
microalgae growth rate remained unaffected.
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* Corresponding author. Tel.: +351 966471036; fax: +351 218317010.

E-mail address: lmredondo@deea.isel.ipl.pt (Luis Redondo).
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1. Introduction

Humanity has a growing need for energy, food, feed and medicines, which resulted in an extensive
demand for fossil fuels, anthropogenic chemical products, land and animal stocks. All these
contributed to a huge pollution issue and a decrease of animal and vegetable wildlife. In order to
mitigate this problem, in the last decade considerable attention has been drawn to water resources

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and specifically to microalgae [1-2].
Microalgae have been used for many centuries as a source of food (eg. Nostoc in China) [3].

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However, it was only a few decades ago that microalgae were looked at with a much higher potential
for the biotechnology market. Since then, microalgae industry has multiplied in different applications

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for food, feed, aquaculture, cosmetics, energy and others [4].
Microalgae are photosynthetic unicellular microorganisms, living in saline or freshwater

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environments that convert sunlight, water and carbon dioxide to algal biomass and oxygen.
Microalgae photosynthesis is today a widely used process for anthropogenic CO2 capture and
sequestration. In fact, photosynthesis is a known method to capture anthropogenic CO2, and aquatic

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microalgae are among the fastest growing photosynthetic organisms, having carbon fixation rates an
order of magnitude higher than those of land plants, as microalgae utilize CO2 as one of the main
building blocks for their biomass [1-2].
In addition, microalgae cultivation allows for production of biomass containing a variety of products
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for a high value market, such as amino-acids, anti-oxidants, vitamins, fatty acids or carbohydrates.
This biorefinery industry attracts investment because of its potential but also demands high
productivity and quality levels in microalgae cultures on a production scale [3-4].
To accomplish this on an industrial scale, it is essential to have strict control of the whole process.
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This reflects a control of physicochemical properties (pH, conductivity, temperature, nutrients, CO2
rate, etc.) but also demands a close control of biological properties, namely biological
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contaminations of the cultures [3]. Specifically, biological contamination of microalgae cultures with
predators such as protozoa can highly jeopardize productivity [14], leading, in extreme cases, to the
total loss of the culture. For this reason, it is of key importance to develop effective tools for the
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selective elimination of these contaminants, without interfering with the normal development of
microalgae.
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The application of Pulsed Electric Field, PEF, which can cause the permeabilization of biological cell
membranes is not a new technique, however nowadays has left the laboratory and is being used
industrially, where it is mainly applied to enhance the extraction of vegetable cells inner contents in
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food industry processes, or as a sterilization method for animal cells [5-6, 8-9]. This phenomenon
known as Electroporation depends on the local transmembrane potential on the cell membrane,
where for a given pulse duration and shape, a specific transmembrane voltage threshold exists,
leading to a specific electric field magnitude threshold for electroporation (Eth). This means that if
E≧Eth the cell membrane gets electroporated [8].
In addition, the effectiveness of the field strength varies with the cell radius, meaning that the lower
the cell radius, the higher the field strength in order to produce similar effects to bigger cell radius.
Hence, this property can be used as a selective tool to process cells with different sizes [6, 8].
The application of Pulsed Electric Fields in microalgae cultures, in laboratory and industrial scale, is
not new. In effect, the extraction of lipids from microalgae, using Pulsed Electric Field, is a huge
research field with industrial application already, as for other intracellular products, such as proteins
and chemicals [10]. However, scarce information exists regarding the use of pulsed electric fields in
order to control contaminants in microalgae cultures. Nevertheless, this technology appears to be
very suitable to implement on an industrial scale, since it can be used selectively to impact
contaminants and not microalgae in production, it does not add or remove any chemical compound
to the cultures and it does not require any significant changes on the production line.
In this work a 10 kV/3.5 kW monopolar pulse solid-state modulator, EPULSUS-PM1-10 from
EnergyPulse Systems, developed for industrial environment, is incorporated on the production scale
of unicellular microalgae with the aim of: a) understanding the effects of PEF in microalgae culture;
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b) verify the size selectivity of PEF in the different cells existent in the microalgae culture; c) verify
the effect of PEF in reducing the number of protozoan contaminants, without affecting the viability
of the culture [11].

2. Material and methods

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2.1. Microalgae Culture

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The microalgae culture consists of an industrial contaminated Chlorella sp. culture. Chlorella sp. cells
have a spherical shape with about 5 µm in diameter. Microalgae were grown, with an industrial

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complex medium, in horizontal closed tubular (56 mm tube diameter) photobioreactors (PBRs) with
2.7 m3 of culture volume (Fig. 1), in a prototype microalgae production unit for CO2 capture and

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biomass production in Pataias, Portugal, developed by a partnership between A4F, AlgaFuel (a
biotech company) and Secil/CMP (a cement company).

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Figure 1. Microalgae photobioreactor installation.

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The culture tubes have about 1100 m, where the microalgae circulate with an average velocity of 1
m/s or 7.9 m3/h. This means that probabilistically it takes about 20 min for one cell to go through the
entire PBR.
The culture was contaminated with protozoa, namely rotifers, generally with several 100s µm in
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length and exhibited an electric conductivity of 2.12 mS/cm. At the start of the tests, the population
of rotifers represented about 4% of cells count on the culture and the presence of rotifer eggs was
almost nonexistent.

2.2. Modulator and transducer

In order to conduct the treatment, a commercial EPULSUS®-PM1-10, 10kV/ 3.5 kW solid-state pulse
modulator was chosen. This equipment, shown in Fig. 2, has only 800x600x400 mm 3 in volume and
80 kg weight so it can be placed in the most suitable location for the treatment, and the operator can
set the PEF protocol in the modulator via a touch screen display, which shows also the essential
process parameters.
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Figure 2. 10 kV/3.5 kW monopolar pulse solid-state modulator, EPULSUS®-PM1-10 from EnergyPulse

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Systems.

The modulator is based on a capacitive discharge from a solid-state Marx type generator, of which a
simplified circuit is shown in Fig. 3, and detailed discussion elsewhere [12].

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Figure 3. Simplified circuit of the solid-state based Marx type generator used in the modulator for producing the
high-voltage pulses.
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One of the advantages of using a capacitive type discharge pulse modulator is the weak dependence
of the voltage waveform on the impedance of the load, as long as the energy stored in the modulator
(i.e. in the electric field of the capacitors) is higher compared to the energy delivered in each pulse,
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normally 5 to 10 times greater, in order to have a voltage droop in the voltage pulse lower than 10 %
[13].
Fig. 4 shows a typical voltage and current pulse waveform from the Fig. 2 modulator; 10 kV / 100 A
pulse, 24 µs width and 100 Hz repetition rate, considering a resistive type load, which is the typical
condition when processing the microalgae, due to the low conductivity of the medium.
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Figure 4. Typical voltage and current waveform delivered to a resistive type load, where (dark trace) voltage
pulse 2 kV/div and (light trace) current pulse 20 A/div from the modulator, with 5µs/div.

The modulator was connected to a co-field treating cylindrical chamber, with 56 mm diameter and

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two 40 mm width gaps, as shown in Fig. 5, through a high-voltage 3 m coaxial cable. The size of the
chamber was defined by the industrial standard tubes installed. The PEF co-field transducer was
installed on the PBR in series, without changing the culture flow and without the need to stop
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production to apply the treatment.
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Figure 5. Simplified schematic of the co-field transducer used for the PEF treatment. In reality the edges are
rounded to decrease the field enhancement.

2.3. Experimental procedure

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The transducer was introduced in the culture tubes on one curve of the PBR, as shown in Fig. 6.

Figure 6. Position of the PEF transducer in the PBR.

The culture was treated for 6 hours with typical pulses of 5 kV and 100 A (i.e. two simultaneous 50 A
pulses into each gap), with 50 Hz and 65 µs pulse width, and approximately 8.5 % voltage droop,
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resulting in an average pulse energy of 2x16.25 J per pulse (i.e. 13 kJ/kg in total, calculated from total
amount of energy delivered in 6 hours, divided by the amount of liquid) and average output power of
1625 W. Working with recirculation, all the culture was uniformly exposed to the PEF throughout the
assay. Considering the flow and electrical parameters, on average, every cell in the culture was
treated with 2x18=36 pulses at the end of the 6 hours test.
Considering the treatment chamber configuration used, shown in Fig. 5, it was calculated from

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simulation that 95% of the volume in the chamber holds an average 900 V/cm electric field, where
due to edge effect the maximum fields at the electrodes is 1650 V/cm and the minimum field in the

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middle of the gap between the high-voltage and ground electrodes is 600 V/cm.
The culture showed a mixed population of microalgae, as well as various contaminants, mainly

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rotifers, but also fungi, ciliates and bacteria, as a result of a forced contamination period preceding
the test. Fig. 7 shows the initial control sample, containing the large rotifers and the smaller

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microalgae.
During the test period, samples were taken from the culture every 30 min and observed under a
microscope (Motic trinocular BA300 1150), in order to view the contaminants activity and microalgae

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structure and viability.
Additionally, the culture was monitored during the subsequent days in order to determine the effect
of PEF on the viability of the culture. Cell concentration was followed by measuring the optical
density (OD, Genesys™ 10S UV-Vis Spectrophotometer) of the culture and the microscopic evolution
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of the population.
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Figure 7. Micrograph of the control culture sample, with three rotifers. Each rotifer measured around 100 m in
length and 50 m in width.

Extraction of the concentrated cells with an organic solvent (acetone, 99.9% AnalaR Normapur®) was
performed to quantify the effect of PEF on the membrane of microalgae. If the membrane is
damaged, the intracellular compounds are more easily extracted by an organic solvent. Microalgae
culture was centrifuged (4000 rpm, 5 min, Nahita model 2655), acetone (3 mL for each mL of culture
sample) was added to the biomass pellet and after 5 min extraction on a vortex mixer the mixture
was centrifuged. The supernatant acetone with the extracted intracellular compounds was measured
by spectrophotometry (Genesys™ 10S UV-Vis Spectrophotometer), quantifying the chlorophyll in the
extract (absorbance at 433 nm), giving an indirect measurement of the intracellular compounds
released.

3. Results and discussion

The results presented here indicate the cumulative effect of exposing the culture to a single pulsed
electric field protocol; in effect, no study was done regarding the effect of different parameters on
the culture.
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The initial temperature of the culture was 22 ºC (the test was conducted in May 2012) and it was
decided that the temperature should not raise more than 5 ºC, which could affect the viability of the
microalgae. At the end of the test, the measured culture temperature was 25 ºC, which was
attributed to the increase of the ambient temperature and sunlight exposure, because the assay was
performed during the day with a maximum ambient temperature of 30 ºC. However, due to the kJ/kg
delivered by the pulses, this increase of temperature could also be due to the application of the PEF.

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During the test, a continued reduction of the normal activity of the rotifers was observed, where its
overall structure was generally retained. Occasionally, rotifers with disrupted and damaged external

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membranes were observed, as shown in Fig. 8. By the end of the test period, 6 hours, only
intracellular rotifer activity was detected, so rotifer motility had apparently been affected.

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Considering the measured temperatures, at the beginning and end of the test, and the 3 ºC
temperature difference, is was concluded that the PEF parameters used had no significant effect on

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the temperature increase of the culture.
After the test, the culture was monitored daily in order to control the evolution of the population of
rotifers. It was observed that the rotifer population gradually decreased until it was almost

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completely washed out after one week, through only the daily renewal of the culture. The population
of rotifers decreased to about 0.5% of cells count on the culture and was mainly composed of rotifer
eggs, indicating cell rupture and distress.
Thus, it is possible to assume that rotifers growth, after exposure to the electric field, was affected,
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since one would anticipate their rapid overpowering of the culture. This conclusion is reinforced by
the fact that during the following two days, no culture renewal was done, which would further allow
the population of rotifers to recover. In fact, precisely the opposite was observed, as the rotifer
population declined.
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Figure 8. Micrograph of the culture sample after 6 hours PEF, with one rotifer. Each rotifer measured around
100 m in length and 50 m in width.

In order to understand the effect of the electric field on the microalgae, samples taken during the
test were processed by extraction with acetone and chlorophyll was quantified. From all the samples,
only in the last sample, after 6 hours of exposure to pulsed electric fields, a peak in absorbance with
twice the value of the other samples was observed, pointing to the extraction of higher amount of
intracellular compounds released from the cells, which may be explained by the weakening/partial
rupture of microalgae cell membranes.
The viability of the microalgae culture was also verified by measuring the optical density (OD) of the
culture. At the day of the test OD was 7.8 and after one week of daily 20 % culture renewals, the OD
measured was 8.9, showing the cultures growing, hence microalgae viability. The OD not only
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stabilized while the culture was being harvested daily, but in fact the culture concentration
increased.
On the whole, considering the pulsed electric field protocol applied, the results show a reduction of
active protozoan population of 87% after 6 hours treatment and 100% after few days of normal
cultivation regime. At the same time, microalgae growth rate increased, supported by the reduction
of rotifer grazing and also showing that they had not suffered any irreversible damage by the applied

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electric field. This selectivity is believed to be related with the significant size difference between
microalgae and protozoa and also with different cell membrane structure between them.

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Conclusions
A unique Pulsed Electric Field protocol was applied during 6 hours to a microalgae culture

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contaminated with rotifers. The results obtained showed that the exposure of the culture to an
electric field causes, in rotifers, visible damage in some individuals (ruptured or damaged
membrane), and mainly functional damage which, although not observable under a microscope,

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caused a decrease or complete loss of reproductive capacity of these organisms and subsequent
washing-out through renewal of the culture.
In relation to the effect on the microalgae, it was found that the application of an electric field
caused changes in cell membranes only after 6 hours. However, the effects are observable only
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during the extraction with acetone (additional stress factor). It is suspected that, with this specific
protocol, these effects are reversible, i.e. cells can regenerate after the effect of PEF, regaining
normal morphology and maintaining viability. Supporting this point, the culture maintained its
growth with daily renewals of 20%.
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It is not possible to conclude whether the effects observed in culture are immediate or cumulative,
because, for rotifers, the results were only confirmed after a couple of days; for microalgae, only
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after 6 hours of treatment it was possible to find any differences based on the extraction with the
organic solvent.
It is possible to conclude that the application of an electric field on a microalgae culture
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contaminated by predators has beneficial consequences for the culture, acting selectively on various
organisms according to their size and membrane properties: inflicting damage to the population of
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rotifers and causing no visible changes to microalgae at the same time.

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article 276, 2014. MA
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Highlights

 We have answer to all questions of reviewer one, as described in the response letter and
highlighted in the manuscript revised;
 We have answer to all questions of reviewer two, as described in the response letter and
highlighted in the manuscript revised;

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 We have revised the English in the manuscript , as highlighted in the manuscript revised

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