Accepted Manuscript

Microalgal biofilms: A further step over current microalgal

cultivation techniques

Antonia Mantzorou, Filippos Ververidis

PII: S0048-9697(18)33844-0
DOI: doi:10.1016/j.scitotenv.2018.09.355
Reference: STOTEN 28859
To appear in: Science of the Total Environment
Received date: 6 July 2018
Revised date: 24 September 2018
Accepted date: 28 September 2018

Please cite this article as: Antonia Mantzorou, Filippos Ververidis , Microalgal biofilms:
A further step over current microalgal cultivation techniques. Stoten (2018), doi:10.1016/
j.scitotenv.2018.09.355

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 ACCEPTED MANUSCRIPT

Microalgal biofilms: A further step over current microalgal cultivation techniques

Antonia Mantzorou and Filippos Ververidis*

Plant Biochemistry and Biotechnology Group, Biological and Biotechnological

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Applications Laboratory, Department of Agriculture, School of Agriculture, Food and

Nutrition, Technological Educational Institute of Crete, Heraklion, Greece

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*Corresponding author
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Technological Educational Institute of Crete, P. O. Box 1939, GR-710 04 Heraklion,
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Crete, Greece.

E-mail: ververidis@teicrete.gr
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Highlights

 Currently, microalgal cultivation in large scale has been limited.

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 Biofilms are different life forms where microorganisms grow attached to substrata.
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 Biofilms could resolve the bottlenecks of suspended cultivation systems.

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Abstract

The scientific community has turned its interest to microalgae lately, because of

their countless applications such as wastewater treatment and pharmaceutical industry.

Nevertheless, so far applied cultivation methods are still prohibitive. Ordinary

cultivation techniques in which microalgae are suspended in liquid medium suffer from

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many bottlenecks, such as low biomass productivities, difficulty in biomass harvesting

and recovery, high installation and operating cost, high water requirements etc.

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Although, microalgal biofilms are known to be a nuisance because of surfaces fouling,

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they have emerged as an innovative technology with which microalgae are developed

attached to a solid surface. This technique seems to be advantageous as compared to

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conventional cultivation systems. Microalgal biofilm systems could resolve the
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problematic aspects of ordinary cultivation techniques such as low biomass

productivities, water management and biomass recovery. A detailed description of this

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technique with respect to the parameters affecting them is reviewed in this work.
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Key words: microalgae, suspended cultivation systems, biofilms.

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1. Introduction
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The considerable potential of microalgae for biomass production and co–

products has been extensively examined lately from many scientific aspects of research

(Berner et al., 2015). Although microalgae seem to be organisms with a range of quite

different applications, there is a need in boosting their quantity and at the same time,

keeping the cost at low levels (Mantzorou et al., 2018). Until now, the microalgal

cultivation in large scale is not quite popular as it cannot become economically viable

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due to high installation and operating costs as well as low productivities (Berner et al.,

2015; Gross et al., 2013). Most studies related to microalgal cultivation, have focused in

suspended cultivation systems (Toninelli et al., 2016).

Until now, there is an significant research aiming to improve the currently used

cultivation systems by either improving process design or increasing biomass yield

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(Berner et al., 2015). Attached cultivation systems based on biofilms seem to be an

attractive process because such systems have less water and energy requirements

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(Ozkan et al., 2012). Microalgal biofilms have been studied from both technological and

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ecological aspects (Zippel et al., 2007). Applications of these biofilms, such as

aquaculture, wastewater treatment and improvement of antifouling substances, have

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gained the scientific interest (Roeselers et al., 2007). Nevertheless, as compared to
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bacteria involvement to fouling, knowledge about microalgae fouling is rather limited

(Zippel et al., 2007).

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The purpose of this work is to revise the current literature concerning microalgal
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biofilms development as a new technology. This review attempts to present the current
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status of existing research data showing the important factors and synergistic effects of

biofilm creation either oriented to marine biofilms or to freshwater systems. Taking into
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consideration of the latter’s scarcity for human consumption, it is obvious that the
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cultivation of marine microalgal species is more encouraged than the freshwater

microalgal cultivation (Khan et al., 2018). Experimental biofilm cultivation systems are

described below with respect of their selected parameters, to give an insight of the

contemporary trends in improving microalgae production. Factors affecting their

performance are analyzed below for identifying the gaps of the ongoing research.

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2. Microalgae

Microalgae are normally unicellular microorganisms possessing a capacity to

aggregate and thus being able to form different types of cell organization, such as

unicellular, colonial and filamentous (Arad and Richmond, 2007; Palma et al., 2017).

They are considered being the earliest life–forms in the earth (Hamed, 2016). They are

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thallophytes i.e. they are plants without leaves, roots or stems and chlorophyll a is the

main photosynthetic pigment (Dragone et al., 2010). Their photosynthetic mechanism is

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similar to terrestrial plants but because of their simpler cell structures and their easiness

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of accessing to CO2 and nutrients, they have greater efficiency in converting solar

energy into biomass (Priyadarshani et al., 2012). They live in a variety of environmental
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conditions and they are found in both aquatic and terrestrial environments. They grow
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under a broad range of temperature and their raise could be confined because of limited

nutrient and light supply (Alam et al., 2015; Hannon et al., 2010). Those tiny
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microorganisms are of high importance due to their influence in the various existing
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ecosystems (Katarzyna et al., 2015; Sevda et al., 2017). As primary producers of the
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food chain, they are the most important level in the maintenance of trophic equilibrium

(Monteiro et al., 2011).

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Microalgae have gained the scientific interest lately, since they have been
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characterized as a raw material for processing or manufacturing industry (Gross et al.,

2013; Katarzyna et al., 2015). The applications of these microorganisms seem to be

countless. Some of them are the fourth-generation biofuels, fertilizers, aquaculture feed,

nutraceutical and wastewater treatment (Gross et al., 2013; Irving and Allen, 2011;

Katarzyna et al., 2015; Zippel et al., 2007). Furthermore, because of their miscellaneous

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environmental habitats, microalgae can be used in ecotoxicological tests, for monitoring

of water quality (Campanella et al., 2001; Garbowski et al., 2017).

The cultivation of microalgae is preferable as compared with the terrestrial

plants due to the reasons mentioned below. I) They have variable chemical

compositions which are dependent on their cultivation medium. This is a result of

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ensuing from their huge biomass biodiversity (Choudhary et al., 2017). II) They do not

compete with the terrestrial plants for agricultural land as they do not need any of it for

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their cultivation (Johnson and Wen, 2010). III) Their nutrients and water requirements

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can be met with the use of wastewater (Garbowski et al., 2017; Zhang et al., 2018). IV)

Their cultivation has no seasonal limitations and some species survive in extreme
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environmental conditions. The fact that they double their biomass in a few hours results
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in an important rise in yields (Sevda et al., 2017; Wu et al., 2012). V) They cause less

environmental pollution because of their less need for pesticides and fertilizers (Das,
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2015). VI) Also, they could remove pollutants (such as nitrogen, phosphorus and heavy
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metals) from liquid waste streams and CO2 from the atmosphere, providing the extra
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benefit of phycoremediation (Hu et al., 2008; Palma et al., 2017; Wu et al., 2012). VII)

Some microalgal species produce greater amount of bioenergy per area than the
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conventional oil crops do (Schenk et al., 2008; Schnurr et al., 2013; Shen et al., 2018).
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3. Conventional cultivation techniques and recovery of microalgal biomass

Currently, microalgae are cultivated in open systems or in closed systems

(Photobioreactors−PBRs). These two system types could have various configurations

(Fig1). In both cases, the microalgal cells are suspended in liquid culture media or water

with the latter being the basic component of the microalgal cultures (Berner et al., 2015;

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Cheng et al., 2013; Liu et al., 2013; Ozkan et al., 2012). It has been calculated that to

produce 1Kg dry biomass of microalgae, about 12–2000Kg of water are needed (Berner

et al., 2015). Both systems are compared in Table 1.

A major problem in production of microalgae in large scale is the harvesting

techniques. Because of their fast growth, their repeated harvesting is recommended

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otherwise photosynthesis will be limited (Garbowski et al., 2017). Furthermore, due to

the small size of microalgal cells (more than a few micrometers), recovery of the

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biomass becomes difficult and costly (Garbowski et al., 2017; Irving and Allen, 2011;

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Lee et al., 2014). Many authors have reported that harvesting techniques is the

determinant factor for the cost assessment of microalgal cultivation (Garbowski et al.,
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2017; Irving and Allen, 2011; Lee et al., 2014; Miranda et al., 2017; Shah et al., 2014).
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Among them, the most popular harvesting techniques are centrifugation, flocculation,

flotation, sedimentation and filtration (Garbowski et al., 2017). The cost of such
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methods accounts for the 30% of the total expense of microalgal cultivation (Irving and
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Allen, 2011; Lee et al., 2014). The proper harvesting technique, which one may choose,
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is contingent on microalgae features such as culture density, cell size and production

purpose (Brennan and Owende, 2010). Nevertheless, none of them has been
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characterized economical and appropriate for microalgal production in large scale

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(Muñoz et al., 2009). These techniques are compared in Table 2.

4. Microalgal biofilms

Biofilm could be characterized as consortium of microorganisms, embedded in

extracellular polymeric substances (EPSs), forming a complex structure which is

developed in solid surfaces (substrata) (Mantzorou et al., 2018). Biofilms are an

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alternative approach of microorganism communities which have totally different

characteristics than those of suspended cultures (Berner et al., 2015).

Despite the limited research, biofilms are not a new phenomenon. The earliest

fossil biofilm that has been recorded, dates 3.5 billion years ago (Roeselers et al., 2008).

Nevertheless, Costerton and his coworkers introduced the term biofilm in order to

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describe a consortium of organisms design, surrounded of extracellular matrix and

attached to a substratum in contact with a liquid medium (Polizzi et al., 2017). In the

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aquatic environment, they cover most of the lower solid layers such as ships, rocks,

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marine animals etc., (Krishnan et al., 2017). They are usually composed with protozoa,

bacteria, larvae and microalgae (Katarzyna et al., 2015). In some cases, it is possible to
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include higher organisms such as macroalgae (Berner et al., 2015). Biofilms, composed
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of multiple species are often used in wastewater treatment while biofilms composed

only of single species are used in manufacturing process (such as biotransformations,

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fermentations etc.) (Li et al., 2007). Biofilms can be found in nearly every liquid–solid
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interface existing in nature and play a crucial role in ecosystems function (Li et al.,
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2007). They contribute considerably to nutrient movement or exchange but also to the

energy flow (Zippel and Neu, 2005).

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Many scientific fields such as engineering, ecology, architecture and biology

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have investigated biofilms (Berner et al., 2015). Biofouling is the phenomenon by which

all natural and artificial submersed surfaces are colonized by a consecution of organisms

(Mieszkin et al., 2013). Until recently, biofilms have been characterized as a problem in

fouling surfaces as they cause alloys and metal corrosion (Irving and Allen, 2011;

Krishnan et al., 2017). In addition, they damage systems related to water distribution,

power plants and cooling systems. In the last case, biofilms raise the pressure drop in

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heat exchangers and diminish the thermal efficiency (Sekar et al., 2004). Within several

minutes, this complex phenomenon takes place and the solid surfaces are covered with

organic and inorganic substances. Hence, the physico–chemical features of the surfaces

are modified and subsequently the settlement of further microorganisms such as

protozoa, bacteria etc., is influenced (Krishnan et al., 2017).

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Microalgal biofilms include all types of particular structures that consist of

microalgae and bacteria which are the main organisms that compose them. These

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biofilms could be found in solid substrata adequately humidified, illuminated and

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capable to supply the microorganisms with nutrients. Many other organisms could be

included in microalgal biofilms (non–axenic cultures) like bacteria which play a crucial
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role in the biofilm formation (Mantzorou et al., 2018). Microalgal biofilms are also
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identified as autotrophic biofilms consisting of microalgae (including cyanobacteria)

and heterotrophic microorganisms (fungi, bacteria and protozoa) (Choudhary et al.,

2017; Sukačová et al., 2015).

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The formation of microalgal biofilms is considered to be a complicated

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procedure and still not sufficiently understood (Katarzyna et al., 2015; Zeriouh et al.,

2017). It is also believed that the process of biofilm formation and growth is different
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and depended on the species that take part in. Hence, it seems difficult to make a point
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about the potential process for the various types of existing biofilms (Choudhary et al.,

2017). Nevertheless, the process of microalgal biofilm formation can be divided into

two steps. In the first step, the cells initially adhere to the solid substratum through

adsorption in order to form a conditioning film. This step is usually reversible. In the

second step, a second irreversible adhesion follows because of the EPSs production

(Shen et al., 2014b).

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Proteins, cations and organic molecules, concentrated on surfaces, favor the

growth of microorganisms and the formation of such biofilms (Christenson and Sims,

2011). The biofilm further grows by cell division of microorganisms which are involved

in the whole process instead of captivating free suspended particles from the nearby

environment (Katarzyna et al., 2015). The biofilm gradually matures and its height raise

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creating a three dimensional structure with multiple layers. The cells are immobilized

and cell clusters are formed. Microalgae are fed with nutrients which are moved via

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diffusion (Berner et al., 2015). Organic substances which are produced

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photosynthetically are available to the biofilm structure. These compounds will be

degraded by bacterial communities (Zippel and Neu, 2005). The biofilm maturity
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influences the abundance and the proportion of participating organisms and EPSs
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(Schnurr and Allen, 2015). Afterwards, at a given biofilm thickness, losses (due to cell

death, grazing, parasitism etc.) become almost equal to the growth. When the loss phase
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overdraws the growth phase, the growth of biofilm structure decreases (Boelee et al.,
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2014b).
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Microalgal cells on biofilm are able to acclimatize themselves in different

environmental conditions (Kesaano and Sims, 2014). Besides, the biofilm formation is
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considered to be a protected growth style which provides to the microorganisms

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advantages concerning their survival in hostile environments (Li et al., 2007; Palma et

al., 2017). It has been shown that bacteria in biofilm forms, resist 100–1000 fold to

antimicrobial substances (Nithya et al., 2014). Guasch and coworkers (2016) examined

the effect of triclosan and snail grazing to diatom community, growing in sandblasted

glasses. They showed that in opposition to the grazer’s presence, under triclosan, the

diatom composition of the biofilm changed. Also, phosphorus uptake capacity of

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microalgae was diminished in the treatments with triclosan whereas the opposite was

observed in the diatoms treated with grazers. In non–axenic conditions, various

organisms can be concentrated in areas which are favorable for them. This leads to the

creation of a heterogeneous biofilm structure. For example, cyanobacteria could be

aggregated at the top of the structure, whereas anaerobic and heterotrophic bacteria

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settle in deeper layers, where oxygen and light are limited (Berner et al., 2015).

The cells of various microorganisms (such as microalgae and bacteria) secrete

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EPSs which help them to be attached and stabilized on the surfaces (Choudhary et al.,

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2017; Zippel et al., 2007). The EPSs are composed with high weight molecules such as

proteins, phospholipids, polysaccharides and nucleic acids and they include functional
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groups such as phosphoric, carboxylic, hydroxyl and amino groups (Christenson and
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Sims, 2011; Shen et al., 2015). EPSs are essential for the biofilm formation because

they keep the biofilm together (Roeselers et al., 2008). Also, they help the
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microorganisms to stay attached to the solid surfaces and the particular matter such as
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erosion products, to be retained in the structure (Bott, 2011; Katarzyna et al., 2015).
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Furthermore, the formed EPS matrix could be considered as a storage space for water

and nutrients and it has a protection role for the cells against extreme environmental
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conditions (e.g. pH and temperature fluctuation, dehydration) and harmful chemicals

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(Berner et al., 2015; Schnurr and Allen, 2015).

EPSs are influenced by various factors such as nutrient availability, stress

response, biofilm age and species composition (Choudhary et al., 2017). It is important

to note that there are microalgal species (such as Chlorella vulgaris) incapable to

produce EPSs by themselves (Katarzyna et al., 2015). It has been reported that the

growth of Chlorella vulgaris alters from planktonic to biofilm when other species are

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present (Irving and Allen, 2011). Nevertheless, (Gross et al., 2013) noticed that

Chlorella vulgaris cells in axenic conditions, did managed to grow attached to a cotton

sheet surface. In addition, it has been shown that the EPS production is enhanced by

increasing light intensity and temperature (Kirsten and Lucas, 2002).

During the last decades, the study of biofilm based cultivation of microalgae has

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gained considerable attention lately (Polizzi et al., 2017; Toninelli et al., 2016). This

interest has recently emerged due to the need of a better and more economically

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harvesting and dewatering algae system (Christenson and Sims, 2011; Gross et al.,

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2013). Microalgal biofilms could be considered as an alternative system for biomass

production. They could resolve the problematic aspects of suspended cultivation

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systems and meet the needs of biofuel production and wastewater treatment (Berner et
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al., 2015; Kesaano and Sims, 2014). Their advantages are mentioned below.

1. The water requirements are less than those of suspended cultures (Podola et al.,
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2016; Shukla et al., 2017). In suspended cultivation systems, for 1 ton of microalgal
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biomass there is a need of 200 tons of water. In contrast, for attached cultivation
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systems, for the same amount of biomass, 17 tons are required for water circulation

which 4 tons of them are used for maintaining surface humidity in appropriate levels
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(Katarzyna et al., 2015).

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2. While the high building cost of ordinary cultivation systems remains a serious

problem for microalgal cultivation, this problem could be eliminated with biofilms.

In the second case, the supporting surface of microalgal cells attachment could be

cheap, easily available and reusable (Garbowski et al., 2017).

3. The biomass productivities of microalgal biofilms are much greater than those of

suspended cultivation systems (Berner et al., 2015; Gao et al., 2015). (Gross and

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Wen, 2014) found that their microalgal biofilm system performance gave in average

302% biomass productivity as compared to the same performance in suspended

cultivation system.

4. Microalgae attached to substrata seem to have high efficiency in wastewater

treatment (Shen et al., 2018). (Ma et al., 2018)(Palma et al., 2017)(Hodges et al.,

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2017)

5. In biofilms, microalgal cells are concentrated in a small footprint area (Choudhary et

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al., 2017).

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6. While harvesting biomass is a nuisance for suspended cultures, this process is

simplified with microalgal biofilms. In the second case, microalgal cells could be
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easily removed from the solid surfaces by scraping (Berner et al., 2015; Shen et al.,
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2014a; Zhang et al., 2018). In addition, the moisture content of biomass is very low

that a further dewatering process is not necessary (Liu et al., 2013). The water
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content of Chlorella sp., harvested by scraping from an attached cultivation system

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was similar to the same species harvested by centrifugation from a suspended

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cultivation system under the same growth conditions (Johnson and Wen, 2010). After

microalgal cells harvesting, the colonies which remain on the substrate will be the
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inoculums for the next growth cycle (Johnson and Wen, 2010).
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7. Microalgae attached to the surfaces have better light availability as compared with

those which are suspended in liquid medium (Katarzyna et al., 2015; Zhang et al.,

2018). (Lee et al., 2014) studied light penetration of attached and suspended culture

systems. They concluded that the sunligh penetration on the upper layer of both

culture systems was almost the same. Howerver, the sunlight intensity on the bottom

of suspended cultivation system was much lower than that of the biofilm systems.

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8. As compared to suspended cultivation systems, biofilm systems can carry out during

short hydraulic retention times (Boelee et al., 2014b).

9. The control of the cell growth area becomes easier when biofilm systems are set up

in lagoons or in the ocean because of the high concentration of the attached cells on

the substratum (Shen et al., 2018).

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Despite the beneficial aspects of microalgal biofilms, the ongoing research

focuses on the potential drawbacks which they may have (Katarzyna et al., 2015). Those

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are referred below.

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1. It is possible for the biomass to be detached from the solid surface. This may lead to

the sedimentation and the loss of pollutants, which might had been bound to the
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biomass, toward the aquatic environment (Garbowski et al., 2017).
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2. As mentioned above, microalgal biofilms through microbial proceeds, cause damage

of metal surfaces such as erosion and decolorization (Katarzyna et al., 2015).

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Until now, a few microalgal biofilm systems have been developed (Table 3).
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Their design and geometric modulation have been chosen according to their purpose
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(Choudhary et al., 2017). However, these systems have been used mainly at a laboratory

scale. Most of them concern the microalgal productivity as compared to the suspended
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cultivation systems. Some of them concern the production of microalgae with the aim of
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treating sewage from various pollutants (Katarzyna et al., 2015; Schnurr et al., 2013). A

few of these efforts are referred below (See section 5).

Nitrogen and phosphorus removal was greater with polyculture microalgal

biofilm in a rotating algae biofilm reactor (RABR) system as compared to the open

pond lagoon (Hodges et al., 2017). Also, BMPBR had better nitrogen removal capacity

than suspended growth membrane photobioreactor (MPBR) did (Gao et al., 2015).

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Although, most studies which are concerned microalgal biofilms are related to nitrogen

and phosphorus removal, their application in wastewater treatment could be considered

as a promising phycoremediation technology with the extra benefit of biomass

manufacturing production for extra bioproducts use (Kesaano and Sims, 2014;

Mantzorou et al., 2018). Ma et al., (2018) showed that the periphytic biofilm cultured in

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different Cu concentrations (2 and 10mg·L−1), had high removal efficiency in Cu (99%).

This is due to the EPS production which offers a great number of functional groups for

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the metal complexation (Palma et al., 2017). (Christenson and Sims, 2012) showed the

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significant potential of a laboratory–scale photorotating biological contactor (PRBC),

established with a microbial consortium dominated by Ulothrix sp., to remove 20–50%

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of toxic metals (such as Cu, Ni, Mn, Zn etc) from synthesized acid mine drainage
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(AMD).

Nevertheless, such systems in wastewater treatment still remain manly in

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experimental stage (Kesaano and Sims, 2014). The design and the large-scale operation
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of such systems become difficult because they are not fully investigated in the so far
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literature (Kesaano and Sims, 2014). Other systems concern the ability of microalgae to

produce biofuels (Ozkan et al., 2012). Some of these constructions are described below.
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5. Main microalgal based biofilm cultivation systems

Based on the liquid medium supply and the biofilm arrangement, microalgal

biofilm designs were first divided by (Berner et al., 2015) to: i) constantly submerged

systems, ii) intermittently submerged systems and iii) perfused systems. According to

his classification, these systems could be categorized into: i) permanently immersed

biofilm i.e. those which are permanently immersed in the liquid culture medium, ii)

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biofilms between two phases i.e. those which are alternated between gaseous and liquid

phases and iii) permeated biofilm systems i.e. those in which culture medium is

provided directly to the substratum (Fig.2). The last categorization could be considered

as more descriptive as far as microalgae and medium arrangement are concerned. In

Table 3, some experimental cultivation systems based on microalgal biofilms are

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described with respect to their selected parameters.

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5.1. Permanently immersed biofilms

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A simple construction was designed by (Shen et al., 2014a) to improve the lipid

yield of Nannochloropsis oculata. In 500 ml beakers, 4 layers of glass fiber–reinforced

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plastic were placed as a supporting material. The beakers were filled with a suitable
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liquid culture medium so as to cover the upper supporting material as well. To examine

oil production, three parameters were tested: nitrogen deficiency, high illumination and
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the combination of the two previous parameters (Table 3a).

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In the study of (Lee et al., 2014) mesh–type materials were placed in open pond
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in order to evaluate the biofilm formation of a microalgae consortium (such as

Chlorella, Nitzschia, Scenedesmus etc). Their performance was compared to another

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one, where microalgal species were suspended in the same open pond (Table 3a).
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An enclosed tubular biofilm PBR was presented by (de Godos et al., 2009). This

system was inoculated with the microalga Chlorella sorokiniana and a mixed bacterial

culture. The system consisted of a 14m transparent PVC tube, arranged horizontally in a

coil manner. Pretreated (centrifuged) swine slurry loading was provided to the system.

The system also included a closed storage reservoir for effluent removal and culture

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recirculation. The enclosed tubular biofilm PBR was evaluated based on the C, NH4+ ,

and PO3−
4 removal from pretreated piggery wastewater.

5.2. Biofilms between two phases

A rotating algal biofilm (RAB) was designed by (Gross et al., 2013). In a

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plexiglass chamber, placed on a rocker shaker, various materials were tested for their

ability as adhesion materials of Chlorella vulgaris cells (Table 3b). This system was

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rotating so that when one corner of the triangle was submerged in the liquid medium,

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the other two corners were exposed to atmospheric conditions. In this way, microalgal

biofilm was exposed both to atmospheric CO2 and nutrients as well. This microalga was
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tested for its biomass yield, biomass productivity and its chemical composition. The
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RAB system performance was compared with the same performance at a flat panel

PBR.
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Another concept was introduced by (Johnson and Wen, 2010). In their effort,
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Chlorella sp. was left to be attached in different supporting materials (Table 3b). Their
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system was composed of a growth chamber in which the tested materials were placed

in. The chamber was gently moved with the help of a rocking shaker. As the shaker was
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moving the chamber, the latter was submerged by half so that when its half was sinked
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on the liquid medium, the other half was exposed to illumination. Their system was

compared with suspended culture which took place on the same chamber without any

supporting material.

A PRBC was designed by (Orandi et al., 2012). For the biofilm establishment, a

microbial consortium, dominated by green microalgae (Ulothrix sp.) was used (Table

3b). For the construction of the PRBC, 16 polyvinyl chloride disks with roughened

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surfaces were used for the biofilm development. The disks placed in a shaft, were

submerged by 40% in a plexiglass tank. The shaft was connecting with a motor for

controlling the rotational speed of the disks. The plexiglass tank was coupled to a feed

container for providing the liquid media (multi–ion synthesized simulated AMD) to the

disks.

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(Blanken et al., 2014) described a PBR devise based on a rotating biological

contactor (RBC). They used Chlorella sorokiniana for evaluating the potential of such

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design in terms of photosynthetic efficiency, productivity and cultivation stability. In

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detail, 4 discs were submerged (by 42%) inside a watertight container. The tested disc

materials were two stainless steel woven meshes (of different tread thickness and
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particle pass size) and one sanded polycarbonate disk (Table 3b).
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5.3. Permeated biofilm systems

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A different biofilm design was described by (Ozkan et al., 2012). Botryococcus

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braunii was cultivated axenically in a biofilm PBR which was composed of a growth
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surface (thick concrete layer) over a wood support plate (Table 3c). The nutrient flow

(which was provided from dripping nozzles above the growth surface) was achieved by
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growth surface tilt.

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Liu et al., (2013) introduced 2 types of attached cultivation bioreactors (Table

3c). The first one which they called it ‘single layer vertical plate’ was composed of a

glass plate, embedded in a glass chamber. While the one plate surface was illuminated,

the other surface was covered with a filter paper, in which the microalgal cells were

attached. The second type of attached cultivation bioreactor was similar to the first. The

only difference was that in the second bioreactor, the glass chamber was composed of

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more than one, glass plates. The glass plates were placed in a way that each of them

received light of different intensity. In this research Scenedesmus obliquus,

Botryococcus braunii, Nanochloropsis OZ-1 and Cylindrotheca fusiformis were

involved. The culture medium was provided by dripping down to the interface of the

glass plate and the filter paper.

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Schnurr et al., (2013) described a semi–continuous system consisting of 12

rectangular flat plates placed in parallel, where the adhesion of Scenedesmus obliquus

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and Nitzschia palea cells was performed on a glass substrate (Table 3c). This system

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was designed for studying neutral lipid productivities under nitrogen and silicon

starvation and growth kinetics. In a similar manner, but using filter paper instead of
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glass, as adhesion material, Cheng et al., (2013) examined the content of Botryococcus
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braunii in lipid and hydrocarbons when the microalga was subjected to nitrogen

sufficiency and deficiency. In both cases, culture medium was provided by flowing
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through the substrate (Table 3c).

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6. Factors affecting microalgal biofilms

The formation and structure of biofilms is contingent on various parameters and

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interactions among living organisms that conduce to settlement and growth (Bott,
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2011). In opposition to bacterial biofilms, there is no much information about the

factors affecting microalgal biofilms (Irving and Allen, 2011). The growth optimization

under various environmental parameters still remains a big challenge. This is a

bottleneck in designing microalgal biofilms for full–scale operation especially in

wastewater treatment (Kesaano and Sims, 2014). Nevertheless, a thorough

understanding of the most influential parameters is crucial for the technical

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development and the design of microalgal biofilms. These information will provide the

tools for the control of such systems and the survival of microorganisms which

participate in (Choudhary et al., 2017).

6.1. Selection of proper microalgal strain

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The choice of the proper strain is probably the most crucial factor in biofilm

formation. Microalgal species have different properties and behaviors. Thus, some

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strains grow better in solid surfaces while others are favored suspended in liquid media

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(Katarzyna et al., 2015). As compared with Scenedesmus obliquus, Nitzschia palea has

been proved to be a very adherent microalgal species with high biomass productivities
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and robust biofilm forms (Schnurr et al., 2013) (Table 3c). Furthermore, some strains
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cannot form single species biofilm, but they should be assisted by other adherent

microorganisms (Li et al., 2007). Irving and Allen, (2011) showed that in axenic
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conditions, between Chlorella vulgaris and Scenedesmus obliquus, the former has lower
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initial attachement (expressed as cells·cm–2) than the latter did, when microalgae left to
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be attached in various materials coupons. It is also a fact that the attachment mechanism

for each organism group differ from one another. The attachement of most diatoms
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proceeds with the EPS production in the form of apical pads, stalks, cell coatings and
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mucilage pads while the attachment of filamentous green algae proceed mainly by the

form of holdfast. Most cyanobacteria attach to the substrata via the EPS production in a

similar form to diatoms and bacteria (Sekar et al., 2004).

6.2. Nutrients

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Nutrient availability is crucial for the microalgal biofilm formation (Katarzyna et

al., 2015). The biofilm growth pattern is characterized from nutrient removal trends. At

the beginning of the growth phase, the nutrient uptake efficiency is relatively low due to

inefficient settlement and adjustment of the biofilm cells. As growth soars, nutrient

uptake increases. Finally, at the death phase, this efficiency is reduced as microalgal

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biofilm loses its integrity because of sloughing (Boelee et al., 2011; Kesaano and Sims,

2014).

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As microalgal biofilms could be considered as different life forms from

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suspended microalgal cultures, there seems to be differences in responding to nutrient

uptakes and deficiencies as compared with suspended cultures (Fields et al., 2014; Shen
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et al., 2014a). This was proved by Schnurr et al., (2013) who studied the potential of
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Scenedesmus obliquus and Nitzschia palea, grown in a biofilm system, for lipid

stimulation under nutrient starvation (Table 3c). They concluded that in contrast to
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suspended growth mode, nutrient depletion did not favor lipid accumulation to
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microalgae. Also, it has been shown that increasing nitrogen concentration results in
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elevated EPS production from green algae species and diatoms (Schnurr and Allen,

2015; Shen et al., 2015). Furthermore, elevated concentrations of nitrogen and

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phosphorus in the surround medium lead to notably higher photosynthetic biomass

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accumulations in a biofilm structure as compared to bacteria (Kebede-westhead et al.,

2003; Villanueva et al., 2011). Under nutrient starvation, EPSs could be degradated in

order for the nutrients enclosed to the matrix, to be used for microalgae feeding or for

the microalgae detaching and repopulation in better nutrient existing conditions

(Schnurr et al., 2013).

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6.3. Light availability

In opposition to bacteria biofilms, microalgal biofilms are strong depended by

light which is the main energy source for their development (Zippel and Neu, 2005). By

a light saturation point (200–400μmol·m–2·s–1), microalgal growth increases in a linear

way with increasing light (Boelee et al., 2014a) (Table 3c). In spite of bioreactor

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configuration or cultivation design, some authors consider light to be the key factor for

microalgal growth optimization (Toninelli et al., 2016). Not only is the adhesion of

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microalgae influenced by light availability but the synthesis and the accumulation of

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EPSs as well (Katarzyna et al., 2015; Zippel et al., 2007). Nevertheless, too much light

in the upper biofilm layers (photoinhibition) or little light in shaded layers

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(photolimitation) can inhibit microalgal biofilm growth (Kesaano and Sims, 2014).
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Concerning lower biofilm layers, in light limited conditions (because of too

thick biofilm or insufficient light intensities), bacteria, EPSs and other non–
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photoautotrophic materials would be dominated (Schnurr and Allen, 2015). It is well

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understood that the microalgal growth is increased as the cells are closed to the light
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source. This was proved by Liu et al., (2013) who examined the growth of Scenedesmus

obliquus in an attached PBR system (Table 3c). They evaluated the biomass
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concentrations at various distances of the cultivation surface from the light source. They
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concluded that the light dimming caused a decreased growth rate of the microalga.

Furthermore, Hill and Larsen, (2005) showed a taxinomic difference among shaded,

unshaded and UV biofilms. The microalgal composition of shaded biofilm was different

from those of unshaded and UV biofilms. Another parameter was tested by Hultberg et

al., (2014) who studied the effects of different light colors on biofilm formation by

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Chlorella vulgaris. They found that white, blue and purple light resulted in sooner and

higher biofilm formation as compared to the treatments with red, yellow and green light.

Changes in light regime could influence the nutrient uptake by microorganisms.

This was confirmed by Sukačová et al., (2015) who examined the phosphorus uptake

from a biofilm composed of a microalgae consortium. During a continuous light regime,

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the efficiency of phosphorus removal reached 97% while the phosphorus uptake ranged

between 36–41% during day–night light regime and using solar radiation. (Hultberg et

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al., 2014).

6.4. Temperature
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Temperature is another factor which could affect species composition,
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microalgal growth rates and grazing activity (Kesaano and Sims, 2014). Increasing

temperature to a point results to a high metabolic activity and therefore to elevated

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biomass productivity (Gross and Wen, 2014). Also, the enzymatic activities by which
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organic matter is degraded by bacteria, are enhanced by increasing temperature

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(Choudhary et al., 2017). Nevertheless, each microalgal species has different

temperature requirements. Generally, concerning mesophilic microalgae, 20–25°C is the

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optimum temperature for maximum growth rates (Boelee et al., 2014a).

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Microalgal biofilm systems are more susceptible to temperature fluctuations

than suspended culture systems because of their considerable low water quantities as

compared to the latter. In the last case, water have a buffer role on temperature (Ozkan

et al., 2012). Furthermore, the water conservation is crucial due to potential evaporation

losses by high temperatures. It has been calculated that evaporation losses of a biofilm

photobioreactor (BPBR) (with characteristic length 10m, water layer thickness 0.05,

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biofilm layer thickness 0.005 and water velocity 0.05m·s−1) in autumn, winter, spring

and summer could reach 3.4, 1.0, 6.0 and 7.3 L·m−2·d−1 respectively (Murphy and

Berberoğlu, 2011; Kesaano and Sims, 2014).

6.5. pH

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Microalgal biofilms depend on pH. Biofilm, as a new micro–environment, has

different pH from the surrounding medium. pH changes are observed among various

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biofilm layers (Katarzyna et al., 2015). Also, pH is crucial for the cell establishment

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(Sekar et al., 2004). EPSs and microalgal membrane are composed mostly of proteins,

polysaccharides and lipids. Different pH could influence the components of

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extracellular metabolites. At low pH values, the dissociation of amine and carboxyl
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groups is enhanced and inhibited respectively. This leads to the weakening of negative

surface charge of microalgae and subsequently to increase of microalgal adhesion.

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Generally, microalgal growth is favored from pH between 6 and 9 (Shen et al., 2014b).
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(Sekar et al., 2004) who examined the effect of pH on microalgae adhesion showed that
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Nitzschia amphibia had significantly better attachment at pH ≥ 7.

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6.6. CO2
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Carbon is essential to photosynthetic microalgae for various metabolisms (Gross

et al., 2013). Carbon is afforded to them from CO2(aq) and bicarbonate (HCO3−) in

wastewater. Atmospheric CO2 and this resulting from organic carbon degradation by

bacteria, are also available to the microalgae (Kesaano and Sims, 2014). Nevertheless,

concerning phototrophic growth, CO2 is considered to be the primary carbon source. If

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the CO2 consumption is more than CO2 supply, the microalgal productivity will be

diminished because of carbon limitation (Blanken et al., 2014).

Concerning suspended culture systems, models have been constructed with

which CO2 loss could be minimal through the mass transfer from the gas to the liquid

phase. On the other hand, while high CO2 levels lead to high productivities, systems

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which could confine CO2 losses have not been thoroughly developed for microalgal

biofilms (Blanken et al., 2017b). However, some systems design (such as RAB system)

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are exposed to the gas phase, and permit to microalgal biofilm to be exposed directly to

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the gaseous CO2 instead of dissolving CO2 in the medium. The increase of CO2

concentration in the inlet air of RAB, beyond the atmospheric levels, did not increase
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more the biomass productivity of Chlorella vulgaris, maybe because of the efficient
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transfer of gaseous CO2 to the microalgal cells (Gross et al., 2013).

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6.7. Attaching material

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Various materials for the microalgal attachment have been tested and compared.
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Among the materials used for this purpose are glass, polystyrene foam, muslin

cheesecloth, polyurethane foam, vermiculite, jute, polyester, cardboard, poly–lactic

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acid, fiberglass, cotton duct, cotton rope etc., (Gross et al., 2013; Johnson and Wen,
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2010; Shen et al., 2014a). The evaluation of these materials is usually based on their

durability and reusability as well as their cost and the degree of cell attachment (Gross

et al., 2013). However, to date, there are no standard materials proposed as biofilm

development substrata (Kesaano and Sims, 2014). Biofilm formation and growth is

affected differently by miscellaneous materials. The properties of them are very

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important for understanding and predict the biofilm development (Schnurr and Allen,

2015).

It has been proven that the texture and roughness of the supporting materials

play a key role in cell adhesion (Katarzyna et al., 2015). In general terms and with only

a few exception results, the cell adhesion is favored of hydrophobic materials (Zeriouh

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et al., 2017). However, there are some who argue that hydrophobicity does not play any

role in microalgae adhesion. Irving and Allen, (2011) showed that the substrate

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hydrophobicity (by means of contact angle) has not been correlated with microalgae

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colonization. Gross et al., (2013) tested a total of 16 materials for attaching Chlorella

vulgaris in a RAB system. Some of them are referred in Table 3b. Cotton duct was
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proved to be the most suitable material because of its durability, low cost and good cell
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attachment. They attribute the suitability of this material to its crevices, which protect

the cells from the sheer force, when they are moving through the liquid. In addition,
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among loofah sponge, cardboard, polyethylene landscape fabric, polystyrene foam,

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nylon sponge and polyurethane foam, polystyrene foam was proved to be the most
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suitable material for the biofilm formation by Chlorella sp., in terms of physical

robustness, biomass harvest, microalgal growth and substratum reusability (Johnson and
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Wen, 2010) (Table 3b).

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6.8. Flow velocity

The biofilm formation is strongly depended on the flow of liquid phase, in

which the biofilm is immersed and supply the microorganisms with nutrients

(Choudhary et al., 2017). The liquid must to circulate with an adequate velocity in order

to supply microorganisms with nutrients and to remove waste products. However, high

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velocity could cause shear stress on biofilm (Berner et al., 2015; Choudhary et al.,

2017). Furthermore, turbulent flows of liquid medium can cause cell detachment and

consequently decrease of biofilm thickness (Berner et al., 2015; Katarzyna et al., 2015).

Johnson and Wen, (2010) noticed that when their attached cultivation system kept

stationary, the microalgal cells settled on the supporting materilal while they could not

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be firmly attached. Nevertheless, at the beginning of cultivation, cell attachment is

favored by a low flow velocity while a higher flow is adequate thereafter in order for the

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microalgae to be provided with nutrients for their further growth (Berner et al., 2015).

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In some configurations, where the microalgal biofilms are settled in a rotating

area alternating them between gaseous and liquid phase, the rotational speed is crucial
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for the cell attachment. A proper speed protects microalgal biofilm from shear stress
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which influences the cell attachment and biofilm thickness (Gross et al., 2013). Johnson

and Wen, (2010) reported that when their attached culture system was kept stationary,
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the cells of Chlorella sp. settled on the substratum without creating a firm biofilm
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structure. They concluded that the settlement of some algae which are naturally found in
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aquatic environments with high current velocities, are favored of rocking which mimics

surge or wave effect. The best rotational speed for biofilm establishment in a PRBCs
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design has proven to be between 1–10rpm (Orandi et al., 2012). Nevertheless, Blanken
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et al., (2014) who examined the influence of disk rotation speeds (3, 6, 11 and 20rpm)

of a RBC based PBR design, on Chlorella sorokiniana growth found that the effect on

biofilm productivities were minimal (Table 3b). Gross et al., (2013) found that in a low

rotation speed (<4rpm) the microalgal biofilm was exposed to the gaseous phase too

long and tended to lose its wetness. On the other hand, at high rotation speed (6rpm) the

biofilm sheared off because of high shear stress (Table 3b).

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6.9. Presence of other microorganisms

Until recently, bacteria were being considered as contamination factor for

microalgal cultures. The last few years this fact has changed. Recent studies have

proved microalgae–bacteria relationship to be essential in algal biotechnology since it

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favors microalgal growth and flocculation procedure (Fuentes et al., 2016). Bacteria

which are present in wastewater have been proven to create a favorable environment on

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which the microalgal biofilm is developed (Schnurr et al., 2013). It is well known that

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microalgae provide O2 which is used by bacteria for the oxidation of NH4 and organic

matter. On the other hand, CO2 is provided by bacterial respiration for the microalgal
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photosynthesis (de Godos et al., 2009). It has been confirmed that in the initial
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colonization process, bacteria strongly interact with microalgae in the biofilm structure.

It has been shown that the biofilm formation in bacteria is associated with Toxin–
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Antitoxin Systems (Karimi et al., 2015). These systems contribute to the processes
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associated with controlling bacterial survival (Wen et al., 2014). As bacterial abundance
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and diversity is higher, more carbon sources are available to microalgae and many more

microalgal cells are attached to the solid surfaces (Choudhary et al., 2017).
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The thickness of Chlorella vulgaris biofilm under non–sterile conditions was

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much greater than those under sterile conditions. On the other hand, Scenedesmus

obliquus formed biofilms with comparable thickness in both sterile and non–sterile

conditions. Nevertheless, in both cases of microalgae, nor S. obliquus or C. vulgaris

managed to dominate in non–sterile conditions (Irving and Allen, 2011). Furthermore,

(Rivas et al., 2010) showed the enhanced and reduced growth of Botryococcus braunii

biofilms under the presence of Rhizobium sp. and Acinetobacter sp. respectively. Also,

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it has been observed that epiphytic bacteria, isolated from Ulva lactuca and Utricularia

reticulata inhibited the growth of Nitzschia paleacea and Cylindrotheca fusiformis.

Furthemore, the cells of Chattonella antiqua and Eucampia zodiacs were lysed by

Alteromonas strains (Mieszkin et al., 2013). Nonetheless, Kawamura et al., (1988)

demostrated that bacteria have no necessarily effect on some microalgae attachment.

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This was proved by Alcaligens sp., wich has no influence on the attachment of Synedra

sp. to substrata.

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Conclusions

Microalgal biofilms is an active research field in early stage. The experimental

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results have been characterized quite promising. These systems, applied mainly in
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laboratory scale, offer valuable information but some points between the factors and the

way that influence the microalgal biofilm development remain ambiguous. Although,
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some efforts have been done, it is necessary such systems to operate in larger scale and
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real conditions for their actual assessment. Different chosen variables hamper the
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assessing of such system performance. For evaluating of their potential, it is necessary

to be assessed using the same baselines of construction and operation.

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AC

Acknowledgements

This research did not receive any specific grant from funding agencies in the

public, commercial, or not-for-profit sectors. All authors declare that there is no conflict

of interest of any kind referring to their work.

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Table 1. Comparison of the most common microalgal cultivation techniques.

Characteristics Reference

Easy structure and operation Garbowski et al., 2017; Lee et al., 2014

Low capital and operation cost Christenson and Sims, 2011; Lee et al., 2014; Sevda et al.,
Advantages

2017

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Easy, combined operation with wastewater treatment Garbowski et al., 2017

Free solar illumination Kumar et al., 2015

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Difficulty in handling contaminations Garbowski et al., 2017; Katarzyna et al., 2015

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Lower biomass productivity than closed systems Christenson and Sims, 2011; Lee et al., 2014

Light limited system Hannon et al., 2010; Lee et al., 2014; Sevda et al., 2017
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Open ponds

High water requirements due to low biomass/water ratio Berner et al., 2015; Sevda et al., 2017
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Cost of water transportation Berner et al., 2015

Disadvantages

Water loss because of evaporation Garbowski et al., 2017; Sevda et al., 2017
D

Difficulty in biomass harvesting Berner et al., 2015; Lee et al., 2014

E

Cost for biomass dewatering Berner et al., 2015

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Dependent on environmental conditions Dragone et al., 2010; Sevda et al., 2017

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Settling in big land area Sevda et al., 2017; Shukla et al., 2017

Insufficient mixing Shukla et al., 2017

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Control of environmental parameters pH, temperature, Kumar et al., 2015; Sevda et al., 2017

light intensity
Advantages

Appropriate for single microalgal species Garbowski et al., 2017; Sevda et al., 2017
Closed systems

Minimized contaminations Hannon et al., 2010

Better light availability Sevda et al., 2017

Disadva

Difficulty in designing bioreactor systems Sevda et al., 2017

ntages

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Low biomass productivity Berner et al., 2015

Difficulty in biomass harvesting Berner et al., 2015; Lee et al., 2014

High water requirements due to low biomass/water ratio Berner et al., 2015

Cost of water transportation Berner et al., 2015

High energy and operation cost Hannon et al., 2010; Sevda et al., 2017

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Potential hydrodynamic stress of culture cells Garbowski et al., 2017

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SC
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MA
E D
PT
CE
AC

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Table 2. Comparison of the most common microalgal harvesting techniques used in

suspended cultivation systems.

Characteristics References

High efficiency of some flocculants e.g. chitosan Suali and Sarbatly, 2012
Advantages

Relatively fast method Al hattab et al., 2015

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Flocculation

Precaution in appropriate selection of flocculants Suali and Sarbatly, 2012

Drawbacks

Chemicals required Brennan and Owende, 2010

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Sensitive to pH changes Shukla et al., 2017

SC
No chemicals required Suali and Sarbatly, 2012
Advantages

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Rapid separation Barros et al., 2015; Dragone et al., 2010

Good biomass recovery Suali and Sarbatly, 2012

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Centrifugation

High energy consumption Barros et al., 2015; Suali and Sarbatly, 2012

Expensive equipment Suali and Sarbatly, 2012

Drawbacks

Inappropriate for large culture volumes Chen et al., 2011

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PT

Possible cell rupture because of shear and gravitational forces Dragone et al., 2010

Cost effective technique Suali and Sarbatly, 2012

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Advantages

No chemicals required Garbowski et al., 2017

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A variety of different membrane designs is available Milledge and Heaven, 2013

Slow process Suali and Sarbatly, 2012

Filtration

Membrane clogging and fouling Barros et al., 2015; Ghosh and Das, 2015; Shukla
Drawbacks

et al., 2017

Need for pre–concentration step Dragone et al., 2010

Appropriate only for large cells Brennan and Owende, 2010

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It does not cause any shear stress to microalgal cells Bosma et al., 2003
Advantages

Non fouling method Brennan and Owende, 2010

Ultrasound

Low operation space Bosma et al., 2003

At this time in progress Suali and Sarbatly, 2012

Drawbacks

Aggregation of other sediments than microalgal cells such as mercury Suali and Sarbatly, 2012

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No chemicals required Brennan and Owende, 2010

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Rapid method Barros et al., 2015
Advantages
Flotation

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Low cost of initial equipment Ghosh and Das, 2015

Low operation space Barros et al., 2015

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Proven to be suitable only for small scale microalgae Al hattab et al., 2015
Drawbacks

Uncertainty about its economical and technical sustainability Brennan and Owende, 2010
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High operational cost Al hattab et al., 2015; Shah et al., 2014

Cost effective Al hattab et al., 2015; Chen et al., 2011

D
Advantages

No chemicals required Chen et al., 2011; Ghosh and Das, 2015

Electrophoresis

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Eco–friendly Al hattab et al., 2015; Chen et al., 2011

Cathode fouling and decrease of current intensity due to its reuse Al hattab et al., 2015
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Drawbacks

Change of cell composition due to high current densities Al hattab et al., 2015
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Table 3. Update of previous published table of experimental cultivation systems, based

on microalgal biofilms. Species are referred to those inoculated and the beginning of the

experiment (Start exp) and to those observed as predominant at the end of the

experiment (End exp). Inoculum is expressed as dry weight of biomass per liter of

culture medium (g DW/L). Illumination is expressed as μmol·s–1·m–2. Where it is

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possible, the hours of the light/dark cycle are also indicated. In some systems, where the

biofilms are settled in a rotating area, the rotation speed is depicted in revolutions per

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minute (rpm). Otherwise it is not specified (–). Furthermore, (–) is used for data that are

SC
neither listed nor displayed in charts at source. Duration is referred to the time interval

(days) between a growth (or regrowth) start up and a subsequent harvest. Biomass
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productivity is expressed as dry weight (g) of the biomass per unit of surface area, per
MA

day (g·m–2·d–1). Here, it is not specified if the mentioned biomass productivity refers to

the initial growth or regrowth modes (subsequent growth cycles). Units are recorded
D

where selected parameters were expressed differently. Some referred results have been
E

calculated and extracted from graphs. Thus, these values are rough estimated (≈). avg.
PT

stands for average. Some parameters differ among the referred experimental setups and

some others are not referred at all. That happens because of the different purpose of
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each experimental setup. Hence, we tried to refer the most appropriate combination.
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Cond. stands for conditions. WW: wastewater.

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Cultivation Biomass
Inoculum Illumination CO2 Duration Flow rate
Culture area Supporting Rotation productivity
Culture system T (°C) (μmol·s–1·m–2) Species Ref
medium material –1 speed (rpm) s
(g DW/L) [light/dark circle (% v/v) (Days) (L·min ) (End exp) –2 –1
(m2) (g·m ·d )
(h)]

T
3a. Permanently immersed biofilms

P
Artificial
seawater (6–
24mM urea)

R I 3.38–3.67

SC
Artificial Glass fiber
Attached culture (Shen et al.,
0.3 seawater (6– 0.02 reinforced 26 2 14 Nannochlorop 1.77–3.87
system 150 [14/10] − − N. oculata 2014a)
24mM nitrate) plastic sis oculata

Artificial

N U
A
seawater (6– 5.32–15.76
24mM glycine)

100 [16/8] blue

M 5.24·10−2

E D
light

100 [16/8] green

P T light
0.81·10−2

E
100 [16/8] yellow 0.52·10−2
4 Chlorella
Glass flask 10 cells light (Hultberg et

C
Z8 medium − Glass 20 − 7 Chlorella
biofilm system mL−1 − − vulgaris, al., 2014)
vulgaris

A C 100 [16/8] red light

100 [16/8] purple

light
bacteria 0.49·10−2

4.76·10−2

100 [16/8] white 5.80·10−2

light

48
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Anabaena
1.8 A. cylindrical 110mg DW
cylindrical
Petri–dish (Kholssi et al.,
BG11 medium 18–28 Wood biochar 18–28 – 20 – –
biofilm 100 [16/8] 2018)
Klebsormidium
1.8 K. flaccidum 43.63mg DW
flaccidum

P
Microalgae
T
Attached growth – Municipal 13.5
Nylon mesh
–
272–520 Natural
conditions – 18
–
−

R Iconsortium
(Chlorella sp,
Pediastrum sp.,
– 9.10
(Lee et al.,
2014)

SC
system WW (sunlight)
Nitzschia sp.,
e.t.c.)

Algal biofilm
membrane PBR
40
Simulated
secondary
0.28
Flexible fiber
bundles
25–28
8000lux
(maximum light 4 20

N U
0.07
−
Chlorella
vulgaris
C. vulgaris 0.05
(Gao et al.,
2015)

A
intensity on the
(BMPBR) effluent
reactor wall)

20ml
Phosphate– 0.01/Petri– Whatman GF/C M Random Indigenous
Filamentous
fungus, (Palma et al.,

D
Petri–dish sub– 24 – 18 ≈0.64
enriched dish filter 134–203 [12/12] – rotation mixed microalgae Chlorella–like 2017)
biofilm system culture
tailing water

E microalgae cells

PT
≈0.11cm–3·m–2·d–
Bacteria, algae 1
10 [16/8]

C E Phototrophic

Algae
≈0.21cm–3·m–2·d–

C 25 [16/8] 1
Modified Polycarbonate biofilm material (Zippel and
Flow–lane – 0.09 20 – 41 −

A
BG11 slides 1.67 from WW Neu, 2005)
incubator
medium ≈0.06cm–3·m–2·d–
Algae 1
85 [16/8] treatment

Algae,
125 [16/8] ≈0.9cm–3·m–2·d–1
cyanobacteria

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Culture
medium (2g·L– 9.27
0.03g
1
urea)
Membrane wet Mixed cellulose Chlorella (Rincon et al.,
1.52·10–3 26 – 8 – C. vulgaris
biofilm reactor biomass esters membrane 50 [14/10] 10–3 vulgaris 2017)
Culture
·L–1

T
–
medium (5g·L 12.64

P
1
urea)

Vertical algal
biofilm (VAB)–
R I
Chlorella
vulgaris, C. vulgaris, O.

SC
Synthetic WW
enhanced Oscillatoria tenuis, S. (Zhang et al.,
– with different 0.16 Coral velvet 28 0.05 8 (6.59– – 10.54–14.68
raceway 425 [14/10] –3 tenuis, obliquus, 2018)
nutrient load 21.5)·10

U
Scenedesmus bacteria
obliquus

A N
3b. Biofilms alternated between gaseous and liquid phase
Scenedesmus

M
S. dimorphus
dimorphus 0.39
(UTEX 417)
(UTEX 417)

E D Chlorella C.
0.11

T
protothecoides protothecoides

Attached algal 106cells· Modified Basal

–1
0.02
E P
Stainless steel
26 − 15
−
8
Chlorella
vulgaris
C. vulgaris 0.04
(Shen et al.,

C
culture system ml medium sheet 100 [24/0] 2014b)
Scenedesmus

C
S. obliqnus 0.18
obliqnus

A S. dimorphus
(FACHB-496)
S. dimorphus
(FACHB-496)
0.32

Chlorococcum Chlorococcum
0.53
sp. sp.

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Photo–rotating Microbial
biological consortium,
Synthesized (Orandi et al.,
contactor 0.1 1.6 PVC 19−23 − 60 2−5 dominated – 0.42
AMD 756 [12/12] 0.01 2012)

(PRBC) by Ulothrix sp.

Cotton rope

P T ≈2.12

Polypropylene

R I 0

Nylon

Polyester
S C 0

≈0.29

U
Bench scale
Mixed culture (Christenson
rotating algal

N
– WW effluent 0.19 Cotton (low 14–24 – 26 4.8 (microalgae and – and Sims,
biofilm reactor 290 [14/10] – ≈1.42
thread) bacteria) 2012)
(RABR)

Cotton (high
thread)
M A ≈1.54

Jute

E D ≈1.62

Acrylic

P T Diatoma sp.,
≈0.85

E
RABR– Mixed culture (Christenson
Enhanced – WW effluent 2.72 Cotton cord 14–24 Natural conditions – 20 5.4 (microalgae, 20 and Sims,

C
– Pediastrum sp.,
Raceway Pond (sunlight) bacteria) 2012)
Chlorella sp.

Pilot Scale
– WW effluent
A
4.26
C Cotton cord 9.6–19.2
208 avg.
– 12 1.2
Mixed culture
(microalgae and
– 31
(Christenson
and Sims,
Natural conditions 11.4 bacteria)
RABR
2012)
(sunlight)

Rotating algae – WW 0.029 20 – 84 – – 4.11

Braid cotton 230 [24/0] Polyculture Filamentous (Hodges et al.,
biofilm reactor

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(RABR) rope microalgae cyanobacteria 2017)

≈7.43
399 [16/8]

≈6
399 [12/2]

T
5
M8a medium Stainless steel Chlorella C. sorokiniana & (Blanken et
Alga disk reactor 0.5 0.06 37 7 9 ≈7.14

P
(30mM urea) mesh 12–635 [16/8] 0.54 sorokiniana bacteria al., 2017a)

399 [24/0]

R I ≈11

Glass fiber–
399 [24/0]
10

S C ≈11

reinforced
plastic

N U 1.47

Plastic film

Silicone film
M A ≈0.63

≈1.1

Maifan stone
sheet

E D ≈1.03

PT
Chlorococcum
Attached algal 106cells· Modified Basal Polyethylene sp. Chlorococcum (Shen et al.,
0.02 26 − 15 8 ≈0.95
culture system ml–1 medium foam 100 [24/0] − sp. 2014b)

C E
Frosted glass ≈0.72

A C Stainless steel
sheet
0.53

Polyurethane
≈0.6
sheet

Polycarbonate
≈0.45
sheet

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Cotton duct 1.08

Laboratory−scal
e rotating algal Bold’s Basal Cotton rag 0.7
Chlorella (Gross et al.,
biofilm (RAB) 4 4.8·10–3 25 – 15 30 C. vulgaris
110−120 [24/0] – vulgaris 2013)
medium Cotton denim 0.09

T
system
Cotton corduroy 0.08

0.33
I P ≈2

R
SC
Laboratory−scal 0.67 ≈2.5
e rotating algal Bold’s Basal 25 −
Chlorella (Gross et al.,
biofilm (RAB) 2 45·10–3 Cotton duct 7 − 2 C. vulgaris ≈2.5
110−120 [24/0]

U
2013)
medium vulgaris
≈3.5

N
system 4

Laboratory−scal
M A
0.03
6 ≈2.2

≈3.5

e rotating algal

biofilm (RAB)
2
Bold’s Basal
45·10–3 Cotton duct 25

E D
110−120 [24/0]
1
7
−
4
Chlorella
vulgaris
C. vulgaris
≈3
(Gross et al.,
2013)

T
medium 2 ≈3

system

E P 3 ≈3.2

C
261 avg. Natural January–
Pilot−scale RAB 22.4 avg. 1.99
conditions February
enhanced
raceway pond
system
–
Bold’s basal
medium

A
14

C Cotton duct

25.5 avg.
(sunlight)
642 avg. Natural
conditions
–

May–June
− −
Chlorella
vulgaris
C. vulgaris

4.29
(Gross et al.,
2013)

(sunlight)
Rotating Stainless steel
Chlorella (Blanken et
biological – M8a medium 0.36 woven rough 38 15mol·m–3 7 11 C. sorokiniana 20.7
422 [24/0] 6 sorokiniana al., 2014)
contactor (RBC) mesh

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Stainless steel
woven smooth 18
mesh

Sanded
14.8
polycarbonate

P T
Algadisk 3

6
R I 18.7

19.5

SC
15mol·m–3
11 20.1

Stainless steel
N U 20

3
18.5

≈1.8
– M8a medium 0.36 woven rough
mesh
38
422 [24/0]
A
0.7mol·m–

M 3
7 6
11
Chlorella
sorokiniana
C. sorokiniana
≈2
(Blanken et
al., 2014)

D
20 ≈1.9

E 3 ≈1.8

PT
0.06mol·
11 ≈2
m–3

C E
Polystyrene
20 ≈1.9

Attached culture Dairy manure A C foam

Cardboard 10
15−60
tips·min–1
Chlorella sp. 2.57

1.47
(Johnson and
0.5 13.6·10–3 20 – − Chlorella sp.
system WW 110–120 [24/0] Wen, 2010)
Polyethylene
0.58
fabric

Loofah sponge 1.28

54
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3c. Permeated biofilm systems

Glass fiber
reinforced 3.19
Modified basal

T
plastic Botryococcus
B.braunii
braunii

P
medium

I
Polyethylene
≈1.65
Multi–layers foam
106cells·
ml–1
1.23 26
100 [12/12]
− 20 3.33 −
R (Shen et al.,
2015)

SC
PBR Glass fiber
reinforced 2.91
plastic B.brauni , other

U
WW B.braunii
microorganisms

N
Polyethylene
2.71
foam

BG11 medium

M A Scenedesmus
obliquus
S. obliquus 10.46

BG11 medium

E D Botryococcus
braunii
B. braunii ≈6.8

Artificial
Cellulose
P T
E
seawater
Single layer – acetate/
enriched (Liu et al.,
10–3

C
vertical plate 25 2 8 – – Nanochloropsis Nanochloropsis
100 [24/0] ≈5.25 2013)
attached PBR nitrate OZ–1 OZ–1

C
with full
membrane
strength BG11
nutrients

Artificial
A Cylindrotheca C.
seawater
≈5.25
enriched with
fusiformis fusiformis
f/2

55
 ACCEPTED MANUSCRIPT

nutrients

≈3.5
12.8 [24/0]

≈4
14.2 [24/0]

T
Cellulose
Attached PBR acetate/ ≈4

P
18.7 [24/0] Scenedesmus (Liu et al.,

I
with multiple – BG11 medium 6·10–3 30 2 9 – – S. obliquus
obliquus 2013)
glass plates nitrate ≈5.6

R
29.3 [24/0]
membrane

56.2 [24/0]

134.5 [24/0]
S C ≈7.8

≈12

0.5g·L–1
fresh
N U Botryococcus (Ozkan et al.,

A
Biofilm PBR BG11 medium 0.28 Concrete slab 25 – 35 0.15 − B. braunii 0.71
inoculu 55 [24/0] braunii 2012)

M
m

D
S. obliquus,
Scenedesmus
PBR system Unsterile bacteria, other 2.1
Semi−continuou secondary WW

E obliquus
microbes

PT
(Schnurr et al.,
s flat plate 0.2−0.3 effluent with 0.14 Glass 26 2 14 23.3 ·10–3 −
80 [16/8] 2013)
parallel synthetic N. palea,

E
horizontal medium Nitzschia palea bacteria, other 2.8
microbes

C C
Polyethylene–
Phototrophic Phormidium
2.7–4.5

A
Phototrophic autumnale,
Municipal WW based Outdoor (Boelee et al.,
biofilm pilot– – 8.08 Natural conditions – June–October 24 – biofilm material Scenedesmus
effluents cond. 2014a)
scale reactor (sunlight) from WW brasiliensis,
woven geotextile
treatment plant Cymbella minuta

Vertical – Synthetic WW 0.13 Polyethylene– 21 10.5 20 0.18 – Microalgal – 2.7

180 [24/0]. (Boelee et al.,
phototrophic based woven mg·L–1 biofilm material

56
 ACCEPTED MANUSCRIPT

biofilm reactor geotextile (avg.) from 2014b)

inorganic
carbon municipal WW
treatment plant

T
Biofilm reactor Cellulose
9g Scenedesmus (Toninelli et
cultivation BG11 medium 0.09 acetate/nitrate 17 1 3 5.06·10–4 − S. dimorphus 9.13

P
–2 200 Flashing light
DW·m dimorphus al., 2016)
chamber membrane

R I Phormidium sp.,

SC
Oscillatoria
Parallel plate
Polycarbonate 113−213 Natural Scenedesmus (Zamalloa et
– Domestic WW 0.5 15−21.7 – 130 1.4 – sp., diatoms, S. 1.92

U
sheet conditions obliquus al., 2013)
reactor obliquus,
(sunlight)

A N bacteria

M
Single layer 0.08
Modified Chu
vertical plate 5.5·10–4 5.5−6.5
13 medium 100 [24/0]

D
attached PBR
Nitrate

Attached PBR
–
0.03/ glass
plate (not
cellulose/cellulos
e acetate

T
25
E 1 10 –
Botryococcus
braunii
B. braunii
(Cheng et al.,
2013)

with multiple
glass plates
Chu 13
medium
identifying
the number
of glass
filter membrane

E P 500 [24/0]
0.01 49.1

plates)

C C Polyurethane

A
Synthetic
Rotary biofilm Scenedesmus
band
reactor –2 obliquus (Travieso et
– with 2% 0.12 27–30 4.6–6kW·m – 20 – 2 – –
(BIOALGA al., 2002)
natural [11/13]
reactor)
sewage

15g·m–2 BG11 medium 1.6 Porous canvas 30 2.5 6 – – ≈21.12

Capillary– 700 [12/12] Desmodesmus Desmodesmus (Shen et al.,

57
 ACCEPTED MANUSCRIPT

effect–based sp. sp. ≈21.85 2018)

812 [12/12]
biofilm reactor
(CBR) ≈17.86
938 [12/12]

≈23.24
1134 [12/12]

938 [24/0]

P T ≈31.54

938 [16/8]

R I 33.92

938 [12/12]

938 [8/16]
S C ≈16.83

≈23.67

700 [24/0] for 2

days & 1134
N U 30.21

[8/16] for 6 days

700 [24/0] for 2
days & 1134
M A
8

27.95

D
[8/16] for 2 days

E
& 938 [16/8] for
4 days)

Lake water PVC

P T Green
microalgae,
Green
microalgae,
Tubular
periphyton
bioreactor
–
with Woods
Hole culture
0.0226

C E30
2500lux [14/10]
– 4.5 0.02·10–3 –
diatoms,
Cyanobacteria
diatoms, bacteria
(such as
Gammaproteoba
–
(Ma et al.,
2018)

C
medium bacteria, fungi,
cteria, fungi,
protozoa

A
protozoa

58
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Fig1. Simplified designs of microalgal suspended cultivation systems: [a] raceway

pond; [b] circular pond; [c] helical PBR; [d] vertical PBR; [e] flat–plate PBR.

Fig2. Simplified designs of some microalgal biofilm systems: [a] System of

permanently immersed biofilms; [b] and [c] biofilms between two phases; [d]

permeated biofilm system.

PT
RI
SC
NU
MA
E D
PT
CE
AC

59
Figure 1
Figure 2