Scholarly article on topic 'Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs'

Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs Academic research paper on "Chemical engineering"

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{Aeroterrestrial / Biofuels / Cultivation / Microalgae / Non-enclosure / Non-suspended}

Abstract of research paper on Chemical engineering, author of scientific article — Ledwoch Katarzyna, Gu Sai, Oinam Avijeet Singh

Abstract Microalgae are getting more interests from industry and science communities. Applications of these small, unicellular microorganisms are countless: from fourth generation biofuels, through fish feed to pharmaceuticals. Ordinary methods of cultivation may be associated with many problems such as high costs, high energy consumption, and low product yield. It is difficult to control contaminations in open ponds while photobioreactors are mainly at laboratory scale and expensive to scale-up. Scientists are investigating various methods of microalgae cultivation and processing to overcome those problems. One of the novel approaches is the non-suspended method for microalgae culturing, where microalgae are grown on attached surfaces. Growing microalgae on surfaces is an attractive option and showing promising results. In comparison with ordinary suspended photobioreactors, the attached systems offer higher biomass yields, easy to scale-up with better light distribution within the reactor and better control of contamination. Moreover, the consumption of water can be drastically reduced. So far, there is not enough research for this method. Limited studies have been reported on enclosure mode of this approach with algae encapsulation into matrix. It is found that this mode would be difficult to scale up due to high costs of the enclosure material and difficulty of separating microalgae from matrix. Non-enclosure mode is more promising way of non-suspended cultivation. So far, no work has been carried out to conduct non-suspended culturing with the use of aeroterrestrial microalgae. They are species growing on the surfaces at highly humid environments. Using them in attached cultivation systems could potentially lower the water consumption to minimum. Studies have shown that the biomass of lower water content can be produced if compared to non-suspended cultivation methods. In addition, mechanization of the cultivation and harvesting processes would be less complex, as the product will not be immersed in the liquid. There would be no need for glass reactors, as lights can be placed in the spaces between surfaces. The light distribution is predicted to be the highest among all existing methods, as there would be no free floating particles absorbing and reflecting light. It will only need humid conditions, rich in CO2 between attachment surfaces. To evaluate potential advantages for non-suspended culturing of aeroterrestrial microalgae in non-enclosure way, proper experiments need to be conducted. In this review, basic concepts of attached cultivation system are discussed, focusing on the studies of biofilm formation including factors affecting deposition and systems. The detailed description of aeroterrestrial microalgae is included to give insight into potential applications of the species into attached cultivation systems.

Academic research paper on topic "Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs"


Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs

Ledwoch Katarzyna, Gu Sai * Oinam Avijeet Singh

School of Engineering, Cranfield University, Bedfordshire MK43 0AL, United Kingdom


Microalgae are getting more interests from industry and science communities. Applications of these small, unicellular microorganisms are countless: from fourth generation biofuels, through fish feed to pharmaceuticals. Ordinary methods of cultivation may be associated with many problems such as high costs, high energy consumption, and low product yield. It is difficult to control contaminations in open ponds while photobioreactors are mainly at laboratory scale and expensive to scale-up. Scientists are investigating various methods of microalgae cultivation and processing to overcome those problems. One of the novel approaches is the non-suspended method for microalgae culturing, where microalgae are grown on attached surfaces.

Growing microalgae on surfaces is an attractive option and showing promising results. In comparison with ordinary suspended photobioreactors, the attached systems offer higher biomass yields, easy to scale-up with better light distribution within the reactor and better control of contamination. Moreover, the consumption of water can be drastically reduced. So far, there is not enough research for this method. Limited studies have been reported on enclosure mode of this approach with algae encapsulation into matrix. It is found that this mode would be difficult to scale up due to high costs of the enclosure material and difficulty of separating microalgae from matrix. Non-enclosure mode is more promising way of non-suspended cultivation.

So far, no work has been carried out to conduct non-suspended culturing with the use of aeroterrestrial microalgae. They are species growing on the surfaces at highly humid environments. Using them in attached cultivation systems could potentially lower the water consumption to minimum. Studies have shown that the biomass of lower water content can be produced if compared to non-suspended cultivation methods. In addition, mechanization of the cultivation and harvesting processes would be less complex, as the product will not be immersed in the liquid. There would be no need for glass reactors, as lights can be placed in the spaces between surfaces. The light distribution is predicted to be the highest among all existing methods, as there would be no free floating particles absorbing and reflecting light. It will only need humid conditions, rich in CO2 between attachment surfaces. To evaluate potential advantages for non-suspended culturing of aeroterrestrial microalgae in non-enclosure way, proper experiments need to be conducted. In this review, basic concepts of attached cultivation system are discussed, focusing on the studies of biofilm formation including factors affecting deposition and systems. The detailed description of aeroterrestrial microalgae is included to give insight into potential applications of the species into attached cultivation systems.

© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license



1. Introduction..............................................................................................................................................................................................................1419

2. Microalgae cultivation: an overview........................................................................................................................................................................1419

2.1. Suspended vs non-suspended cultivation....................................................................................................................................................1419

2.2. Enclosure vs non-enclosure methods..........................................................................................................................................................1420

2.3. Aquatic vs aeroterrestrial microalgae..........................................................................................................................................................1420

* Corresponding author. Tel.: + 44 1234 758207; fax: +44 1234 754685. E-mail address: (G. Sai).

1364-0321/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

journal homepage:



Article history:

Received 9 March 2014

Received in revised form

1 October 2014

Accepted 3 November 2014

Available online 20 November 2014








3. Non-enclosure methods.............................................................................................1420

3.1. Biofilm formation............................................................................................ 1420

3.2. Extracellular polymeric substances (EPS).......................................................................... 1421

3.3. Deposition factors............................................................................................ 1421

3.3.1. Light intensity........................................................................................1421

3.3.2. Nutrient concentration.................................................................................1421

3.3.3. pH..................................................................................................1422

3.3.4. Flow of medium.......................................................................................1422

3.3.5. Strain selection.......................................................................................1422

3.3.6. Substrate properties....................................................................................1422

4. Application of attached systems.......................................................................................1424

4.1. Microalgal biofilms in wastewater treatment....................................................................... 1424

4.2. Microalgae biofilms in biofuels production........................................................................ 1424

4.3. Other experiments............................................................................................ 1425

5. Aeroterrestrial microalgae: research needs..............................................................................1425

6. Conclusions.......................................................................................................1425



1. Introduction

Microalgae are present on the planet Earth from the very beginning of its existence. The interest in these small microorganisms is drastically increasing over the last decades, given their attractive applications in pharmaceutical and many other areas, from simple fish feed to important new generation biofuels. The list also includes specialized medicines, health and beauty cosmetics, fertilizers, and many more The[l,2|.

Scientists have been working to make the production of algae more commercially viable. However, there are many challenges, harvesting is one of them [3]. Processing large volume of microalgae culture is expensive and time-consuming. One possibility is to accumulate microalgae on surfaces during cultivation to allow easy collection. To date, non-enclosure microalgae cultivation has not yet received enough attention. There is no reported study on non-suspended cultivation of aeroterrestrial microalgae. Most of the aeroterrestrial microalgae researchers focus on the problems caused by microalgal biofilm formation, by analyzing mechanisms of attachment and anti-fouling methods of growth control and prevention. This review is to discuss the potentials of using aeroterrestrial microalgae in non-enclosure microalgae cultivation. Literature on the microalgae biofilm formation on artificial substrate is investigated to establish future research routes for microalgae cultivation.

2. Microalgae cultivation: an overview

2.1. Suspended vs non-suspended cultivation

The most common approach in algae cultivation is suspended method, where microalgae are growing suspended in the medium. Algae flows freely inside the container with additional mixing to ensure even distribution of cells. This method shows low concentration of algae grown. The dilution of microalgae in suspended systems is high. Around 99% of culture volume consists of water [4] and only remaining 1% is the dry algal biomass used later on. To obtain dense product, huge biomass are needed to process. Therefore, harvesting large volume of microalgae is extremely expensive process till date [5]. Supplying water to maintain microalgae production is also of high importance. It is projected that about 3800 kg of water is required to obtain 1 kg of biodiesel [6]. Therefore, a huge amount of water is needed for processing microalgal growth in suspended cultivation systems. The

productivity in current suspended systems is low [7]. So far, maximum of few grams of dry biomass per liter of media can be produced during one day of suspended cultivation The[8,9]. The productivity depends on various factors such as microalgae species, reactors and culture density. In the table below there are some selected examples of biomass productivities (Table 1):

At non-suspended mode, algae are grown on surfaces. It leads to accumulation of dense algae inside the reactor. They can be enclosed in the matrix (enclosure method) or form a biofilm on the surface (non-enclosure method) [21]. With non-suspended way of cultivation, it is much easier to separate microalgae biomass from the medium when microalgae accumulate significant quantities of biomass on small area [22]. For harvesting, algae is scratched and dried in the case of non-enclosure method. For

Table 1

Biomass productivities for different kind of suspended cultivations.

Algae specie Productivity [mg/L per day] Type of cultivation Reference

Chlorella sp. 3200 Closed [10]

4025 Open [111

Chlorella vulgaris 40 Closed [12]

136 Mixotrophic [13]

Spirulina platensis 320 Open [14]

320 Mixotrophic [15]

2100 Closed [14]

Botryococcus braunii 26 Closed [16]

155 Closed [17]

Scenedesmus obliquus 140 Closed [18]

150 Closed [17]

Haematococcus pluvialis 76 Open [19]

410 Closed [20]

Table 2

Productivity comparison of suspended and non-suspended cultivation [23].

Species Attached cultivation productivity [g/m2 per day] Suspended cultivation productivity [g/m2 per day] Reference

Scenedesmus obliquus Botryococcus braunii 70.9 5.5-5.7 8.9-14 2.4 [71 The[7,23]

enclosure method, a pre-step is required to extract microalgae from the matrix.

Non-suspended method can be more commercially feasible than ordinary suspended microalgae cultivation. In attached cultivation systems, microalgae are placed on vertically arranged substratum with water supplied only to keep the surfaces wet. Such a system was introduced by Liu in 2013, reaching an average productivity of 70.9 g/m2 per day for Scenedesmus obliquus [7]. In the same reactor, Botryococcus braunii reached productivity of 5.5 g/m2 per day [23]. The production of biomass is given in grams per squared meters in those tests. Both results were compared with ordinary suspended cultivation (Table 2):

Costs associated with water consumption are lower in non-suspended cultivation. To manufacture one ton of microalgae, around 200 metric tons of water are consumed in suspended cultivation [6] whereas in an attached cultivation method, only 17 tons of water is needed for circulation with four tons consumed for surfaces to sustain appropriate wetness level [7]. Till now, there is no attached system operating on big scale for biofuel production, nevertheless promising results are obtained from laboratory scale experiments.

2.2. Enclosure vs non-enclosure methods

The interest in enclosure method of non-suspended microalgae cultivation is growing, as microalgae are easier to control when encapsulated inside the matrix [24]. Experiments on this particular method of cells immobilization are straightforward, given the well-established techniques used for enzymes and organelles entrapment The[25,26]. However, to separate algae from the matrix is not an easy task [21]. The compounds of enclosure may have effects on microalgae species while scaling-up the process would be expensive [27].

In non-encapsulating methods of microalgae culturing, the strain is grown on artificial substrate placed inside liquid medium. Cells have a natural tendency to form biofilm in water habitat. Wild biofilms of different microalgae species can be found commonly. By creating biofilm, it is easier for them to maintain and protect themselves from biocides, predators, and medium conditions (such as pH or temperature). There are a good variety of microalgal species capable of growing on surfaces [7]. They are found on ships, inside reactor tanks or even on the building facades The[28,29]. Biofilms are rich in different species of microorganisms, such as bacteria, fungi, or microalgae [30]. Similar depositions also take place in human organisms, such as blood platelets or dental plaque [31].

Such method has not yet been applied for microalgae harvesting while the only relevant research is on macrofouling [32]. Naturally occurring biological layers have no direct benefits. There are many examples of negative influence of biofilm creation [28], such as pollution for drinking water, reduction of thermal performance for boilers, and possible toxins generated by some algae species [33]. Scientists have been researching for biofouling control and prevention, using biocides, reversal flow, ultraviolet light, and anti-fouling coatings [28]. The knowledge in the field of biofilm prevention needs to be looked at first before starting to use this method for microalgae cultivation.

2.3. Aquatic vs aeroterrestrial microalgae

Microalgae are unicellular microorganisms, widely appearing in aquatic and terrestrial environment. They play a very important role in the ecosystems on the planet Earth. There are a large number of microalgae species with 30,000 species discovered and an estimate of possible 70,000 species [34]. They can be divided according to their taxonomic group or living environment.

Aquatic microalgae are a type of algae naturally occurring inside water reservoirs. All microelements needed by aquatic microalgae sourced from the water. The examples of those strains are Chlorella vulgaris, Scenedesmus obliquus, Pyrocystis lunula, or Nannochloropsis oculata.

Aeroterrestrial microalgae are species growing in biofilms, colonizing both natural and artificial surfaces. They can be found on roof tiles, statues, building facilities, damp rooms, rocks, trees, soil, and many more non-aquatic environments of high humidity The[35,36,37]. They may be found growing together with bacteria, fungi, protozoa, and cyanobacteria The[36,38]. Biofilm thickness can reach up to 0.1 millimeters. Example strains are Klebsormidium sp., Stichococcus sp., Coccomyxa sp., and Apatococcus sp. The [29,35,39,40]. Most of the terrestrial algae can be found in green algae groups, such as Trebouxiophyceae or Chlorophyceae [41]. However, aeroterrestrial microalgae diversity is not well understood [42]. Relevant research mostly focuses on their negative impacts on building facilities. Biofilms causes decolorization and faster weathering of deposited surfaces [36] due to microbial actions triggering breakdown of those materials. Their contribution to the surface weathering is significant, especially that their growth is faster than the growth of higher plants. Species as Gloeothece sp., Chlorella sp., Schizotrix sp., or Chroococcus montanus are good examples of terrestrial microalgae that degrade surfaces The[43,44]. Aquatic microalgae, such as terrestrial ones, are also responsible for surface degradation. They form an unwanted biofilm on ships and industrial tanks. Their biofilms can be found on any artificial surface that is immersed in natural water reservoir for a longer period of time [28].

Aeroterrestrial microalgae have a unique ability to survive desiccation for a long period [45]. They can even survive in a drought when the reproduction is stopped. In comparison with liquid cultures, aeroterrestrial microalgae are flexible and shrink during dry periods (even to around 60%) [46]. Aquatic microalgae such as Nannochloropsis sp. and Scenedesmus dimorphus are not withstanding desiccation well. After drying their growth is significantly limited [47]. In contrast, aeroterrestrial microalgae are easily revived after preservation by drying. It takes only few minutes for Stichococcus sp. and Chlorella luteoviridis to recover photosynthesis after moisturizing [29]. They are highly resistant to hostile environmental conditions as a result, variations in salinity, temperature, and UV variation do not affect them as much as on marine algae [38]. Aeroterrestrial microalgae have a high survival rate at extremely low temperature. Most of 27 aeroterrestrial species tested by Lukesova in 2008 survived cryopreservation, with survival rates above 50% in most cases [48]. There were even species exhibiting 100% of survival rate: all cells of Cylindrocystis brebissoni and Chlorella fusca species survived conservation in extremely low temperature (almost 200 °C).

3. Non-enclosure methods

3.1. Biofilm formation

Biofilms occurring in natural environment are in general created by bacteria, larvae, fungus, protozoa and microalgae The [30,49]. They can be found even in extreme and unfriendly environments, such as nuclear power plants or hydrothermal vents [50]. Formation of microalgae layer is a complex process [28] while the adhesion mechanism is not fully understood The [51,52]. It is believed that the hydrophobic reactions are driving forces for biofilm formation of hard substrates [31].

The first and probably the most important step is the creation of conditioning film The[28,53]. It is a base layer on the surface for microorganisms to grow. There are no clear evidences that the

s O Decreasing rate \ Plateau > Sloughing

Ü) "rö

<D W CO .c Q_ c d) c: o Q. X LU /

Fig. 1. Growth of biofilm in time [28].

conditioning film is required to create biofilm [54], however its formation is essential in promoting cells deposition The[55,56].

Conditioning film formation takes place straight after the surface immersed into the medium, creating the layer consisting of ions and organic molecules [53]. Once conditioning film is formed, microorganisms start their attachment. In the case of saltwater, it takes a few hours for microalgae to attach to the surface [57].

Further growth of the biofilm involves reproduction of microorganisms by division, rather than absorbing free floating particles from the surrounding medium [28]. Before reaching the exponential growth phase, microalgae undergo lag phase (time needed to start reproduction) [58]. At first few days of mixed biofilm formation, microalgae are the microorganisms that dominate the biofilm composition [59], then diatoms start to take over and eventually cyanobacteria become dominant [30].

Growth curve of microalgae in biofilm is similar to that of aquatic algae (Fig. 1). The lag phase is followed by exponential growth, then the rapid development of biofilm stops and reaches the maximum biofilm thickness, at the end the mature biofilm undergoes sloughing.

3.2. Extracellular polymeric substances (EPS)

During biofilm formation, cells produce EPS [60] to create a matrix bonding together the whole biofilm The[28,61]. This creates the environment for growth and reproduction of microorganisms and enables easy attachment of external particles [62]. EPS consists of various groups that function as metal-binding sites. Examples could be negatively charged carboxyl or phosphate group or polysaccharides, proteins, nucleic acids, lipids, phospho-lipids, and humic substances [63].

EPS play an important role in nutrients exchange. They are also responsible for cohesion (binding cells together) and adhesion (binding cells and substratum) [64]. EPS not only act as a nutrient sink, but also protect the whole structure of the biofilm from grazing The[65,66] and action of harmful biocides [67].

Aeroterrestrial microalgae also produce EPS. In addition to the functions above, EPS protect algae from desiccation, retaining water inside algal cells [38] to enable a longer survival during drought. It helps in survival of terrestrial species such as Chlorella trebouxioides, Chlorella luteoviridis, or Stichococcus bacil-laris The[38,44].

3.3. Deposition factors

Various factors contribute to the growth of biofilm with nutrients and lights generally regarded as the most important for microalgae The[28,61]. For aeroterrestrial microalgae, the key factors for growth also include water availability The[29,35,42,68] and chemical/physical properties of the surfaces [53]. The type of surface

is very important, as some attachment surfaces can store the water and the storage ability increases with porous materials [68]. Aeroterrestrial algae prefer rough and porous materials [44] and smaller temperature amplitudes. It is because smaller variation in temperature and presence of cracks keep the surface wet and prevents it from water evaporation The[43,69]. Nutrients concentration is also significant in the case of aeroterrestrial microalgae, however it does not have as much influence on the biofilm composition as other factors mentioned before The[70,71].

Other factors affecting the adhesive strength, amount of biomass formed and its composition could be: disturbance [72], surface roughness The[3,21,73], pH [73], surface rugosity [74], irradiance [75], fluid velocity [76], and concentration of freefloating cells in the medium The[76,77]. More details about their influences on biofilms will be discussed in the following chapters.

To investigate the influence of different factors on biofilm formation, it is important to establish parameters measuring its growth including biofilm thickness, cell counts, or dry mass of formed biofilm [78]. A typical apparatus used for monitoring the biofilm is the "Robbins device" The[28,53]. In Robbins device, test plates are placed inside aluminum block. The liquid is passing through flow channel and the biofilm is formed on test plates. It is possible to remove those plates later and study the accumulation of biomass as well as the influence of liquid velocity. The other techniques for cell biomass or biofilm activity measurement are scanning electron microscopy (SEM), transmission electron microscopy (TEM), scanning controlled laser microscopy, adenosine triphosphate (ATP), total organic carbon (TOC) measurement, light microscopy, and Confocal Scanning Laser MicroscopyThe [28,53,78,79].

All the factors mentioned above have an influence on the development of microalgal biofilm and its composition, however the extent to which they are affected mostly depends on the strain to be attached [21].

3.3.1. Light intensity

The availability of light determines the presence of microalgae in naturally occurring biofilms [28]. The intensity can increase or decrease their adhesion. The attachment is weaker with limited light and generally microalgae growth increases with light intensity [61]. Once the growth reaches its limit, cells undergo photoinhibition and the growth declines.

Light intensity works differently for aeroterrestrial microalgae. The study on Stichococcus and Chlorella luteovirdis species showed [80] that aeroterrestrial microalgae exhibit high tolerance to UVA and UVB radiation. It is due to the presence of mycosporine-like amino acids (MMA), absent in Ulvophyceae or Chlorophyceae group. Aquatic alga, Desmodesmus subspicatus, was affected by too high irradiation by slowing its pace of growth. Some of aquatic microalgae species can stop their growth at all in the presence of UVA and UVB radiation (Table 3) [80]. Great tolerance of aero-terrestrial microalgae to variations of light intensity is very advantageous, as the photoinhibition of cells is a main problem while culturing algae in biofilms [81].

3.3.2. Nutrient concentration

Nutrients are essential for the development of microalgae film. The amount of nutrients should be maintained at a proper level. Above that level, the attachment of cells stops increasing The [55,82]. In mature biofilms, cells closest to substratum surface have limited access to nutrients [28]. It results in their death and sloughing of whole biofilm. The needs for appropriate nitrogen, phosphorous and other elements level strongly depend on micro-algae strain. Some species require extra amount of silica [83]. Addition of glucose to biofilms can enhance the accumulation,

Table 3

The effect of PAR, UVA and UVB radiation on selected algal species [80].


Photosynthetically active radiation (PAR) 50 PPF

Ultraviolet radiation (UV) 8 W/m2 UVA 0.4 W/m2 UVB

Specie Type MMA Growth in PAR + UVA Growth in PAR+UVB Recovery in PAR+UVA Recovery in PAR+UVA/B

Stichococcus sp. Aeroterrestrial Yes No change No change Full Full

Chlorella luteoviridis Aeroterrestrial Yes No change No change Full Full

Myrmecia incise Aeroterrestrial Yes 30% decline 43% decline Full Full

Desmodesmus subspicatus Aquatic No 33% decline Inhibition Full 80%

0.5 1.0 1.5 Water velocity m/s Fig. 2. Dependence of biofilm thickness on water velocity [87].

however the structure formed is loose [76]. Aeroterrestrial algae are able to withstand extreme or harsh conditions, although nutrient rich surfaces are more favorable [36]. The composition of nutrients needed by aeroterrestrial microalgae is the same as in the case of aquatic microalgae, unique for each species.

3.3.3. pH

pH of the cultivation medium affects microalgal growth and biofilm establishment [84]. The structure is influenced by pH even more than by nutrients [39]. It can also happen that the pH within microalgal layer is different from the surrounding medium [76] when microorganisms create a whole new environment separated from the surroundings during biofilming. What is the most favorable pH in the case of microalgae biofilming? The best attachment of Nitzschia amphibian to titanium and glass was obtained approximately at pH neutral environment [73]. This is within the acceptable range given most algae species grow well at pH level from 7 to 9 [85]. The exceptions are green algae found in soils, which prefer acidic conditions [86].

3.3.4. Flow of medium

Movements of surrounding medium influence the biofilm thickness [28] and could be one of the most important factors affecting adhesive strength [76]. Laminar flow, occurring at low velocities, generates thick laminar sub-layer, which enables material to accumulate in dispersed manner [28] and make it easy to remove cells. When the fluid velocity increases, the mass transfer between particles floating in the medium and biofilm increases as well. In the range of 0.6 to 1.6 m/s of fluid velocity, the strength of attachment is improved for Pseudomonas fluorescens. However, the removal of cells from the existing biofilm is also enhanced. An

optimal flow velocity could be found to achieve the maximum growth of microorganism's layer (Fig. 2) [87].

Aeroterrestrial microalgae do not grow within the fluid and the movement of fluid hardly affects them, rather than the presence of water in the form such as rain, highly humid air, fog, or snow [29].

3.3.5. Strain selection

Strain selection is probably the most important for the production of microalgae in non-suspended mode. Microalgae species have different characteristics and behavior. To give insight into differences between microalgal strains, examples are presented below:

- Preferences in way of cultivation: It is evident that some strains may prefer to grow on surfaces while others grow more favorably within the medium as shown by the comparison between Bristles Photobioreactor (PBB) and Bubble Column Photobioreactor (PBC) [88]. It is found that Amphora sp., Navicula sp., and Nitzschia ovalis strains preferred attachment on surfaces with the best results in terms of concentration and biomass yield [88] in PBB (non-suspended cultivation). In contrast, Nitzschia sp. and Cylindrotheca closterium grew better inside ordinary PBC (suspended cultivation).

- Preferences in medium properties: It is reported that Chlorella vulgaris forms thicker biofilm on unsterilized medium and no such observation was found in the case of Scenedesmus obliquus.

- Different predispositions to create biofilms: It was found that not all algal species are capable of producing extracellular polymeric substances (EPS) which affects the quality of biofilms created [89]. An example of such microalgae is Chlorella vulgaris, unable to produce EPS by itself.

- Different influence on attachment surface: For non-suspended culturing, microalgae species may have direct influences on the attachment surfaces. It is found that some microalgae acts as precursors in microbiologically induced corrosion [90] due to the change in pH value and oxygen release during biofilm formation. However, not all algal species induce the biocorrosion of the substrates. It is reported that Porphyridium purpureum does not contribute to steel corrosion [91].

- Strain-specific approaches in attachment improvement: It is possible to stimulate some algal species to accumulate on surfaces. CaCl2 was added to improve Chlorella sorokiniana to form aggregates [92]. Growing Chlorella vulgaris in non-sterile water led to more cells attached to the substratum [89].

3.3.6. Substrate properties

Characteristics of the surfaces are critical for layer formation. According to studies, following factors need to be considered, when designing the process of particle deposition:

- Roughness and texture of surface: They play important role in particles deposition, microalgae grow better on rough surfaces The[3,21,73,74,93,94]. The study of the red algae showed that Halosaccion glandiforme, attached to substrata with features had about 35 times larger density in comparison with density obtained on smooth surface [3]. Also the proper size of substratum dimples can elevate attachment. When dimples are slightly larger than the size of the cells to be deposited, the attachment is higher The[21,95].

- Hydrophobicity: Biofilms are created on hard substrates generally due to hydrophobic reactions [31]. To encourage microorganisms' attachment, it is preferable to have hydrophobic surfaces in particular for saltwater [96].

- Presence of protective layers on surface: Bacteria and diatoms have strong tendency to colonize surfaces, so they can be met even on specially designed antifouling coatings [97]. However, it needs to be kept in mind that microorganisms are less likely to colonize on substratum covered with hydrophilic coating [98]. It is also important to take into account the influence of attachment surface sterilization, as this process changes the properties of the surface [91].

- Costs of surface production: To make non-suspended algae cultivation feasible, the substrate materials need to be cheap and environmental friendly [5]. Surface texturing, desirable in particle deposition enhancement, should be an efficient and not cost consuming process The[5,73].

What are the materials suitable for particle deposition of microalgae? In his work on microalgae attachment, Cui tested a great variety of substrates: teflon, polycarbonate, polypropylene, nylon 6/6, glass microscope slides, and stainless steel 304 [21]. Microalgae tend to accumulate most on the material with the lowest surface free energy, nylon (34.6 ergs/cm2) (Fig. 3). Surface free energy has bigger impact on particle deposition than surface roughness, as stainless steel possessing the roughest surface (124 nm) from all tested materials was not attaching the highest amount of cells [21].

In another study, Sekar conducted experiments with perspex, titanium, stainless steel 316-L, glass, copper, aluminum brass, and admiralty brass. His results showed that the highest attachment took place on stainless steel and titanium (Fig. 4). Remaining materials exhibited weaker promotion of microbial adherence [73].

During studies on rotating algal biofilm system, Gross showed that the most effective material for growing microalgal biofilms for biofuel production is cotton [99]. It was better than other tested materials, such as microfiber, fiberglass, nylon, or vermiculite. The same conclusion was made by Christenson. According to his research, cotton cord was more effective than nylon, polypropylene, acrylic, or jute [100].

Other studies on microorganisms attachment involved poly-dimethylsiloxane, polyimide, polycarbonate plates, silicon, alkane thiolates, plexiglas, and poly-dimethyl silozane elastomer (PDMSe) The[32,74,89,101-107]. All materials above were tested regarding the mechanism of attachment, contact angle data or antifouling properties rather than information which material is the best for growing microalgae in biofilms.

Fig. 3. Growth of Scenedesmus dimorphus and Nannochloropsis oculata on nylon, steel and glass. Surface free energies and surface roughness are given. Data based on work conducted by Cui in 2013 [21].

Fig. 4. Growth of Chlorella vulgaris, Nitzschia amphibia, and Chrococcus minutus on different materials. Data taken from study conducted by Sekar in 2004 [73].

Table 4

Biofilm reactors to treat wastewater with removal efficiencies.

Reactor To clean Removal efficiency [%]

Reactor/culture volume -


Parallel plate microalgae biofilm reactor [112] domestic wastewater 3L+6L 67 96 74 82 - _

Vertical submerged biofilm reactor [109] synthetic wastewater 18L 82.7 - - 98.2 -

Enclosed biofilm tubular reactor [111] swine slurry 7.5L+0.5L 94-100 70-90 - - 61 - 94

Moving bed biofilm reactor [113] raw water from Taihu Lake 45 L - - - - 63.1

Attached algal culture system [115] dairy from wastewater 0.05 L+0.15 L 79 90 - -

TN- total nitrogen TP- total phosphorous COD- chemical oxygen demand TSS- total suspended solids TOC- total organic carbon S2 "- sulfide NH4-N- ammonium

4. Application of attached systems

4.1. Microalgal biofilms in wastewater treatment

There is no much research conducted on microalgal biofilms devoted to biofuels production [108]. Most of the studies are on application of algae biofilms to treat wastewater. Those methods of cleaning wastes have certain advantages. They operate at low temperature and pressure, and there is no requirement for catalyst [109]. In addition, Biofilm processes are not only environmentally friendly treatments, but also effective in terms of procedure expenses The[110,111]. Examples of reactors to treat wastewater with the use of microalgal biofilms are as follows:

- PPMB Reactor: Parallel Plate Microalgae Biofilm Reactor (PPMB) was designed to immobilize nutrients from chemically treated household wastewater [112]. Nitrogen and phosphorous were removed by algal biofilm. The overall removal efficiencies of the system were satisfactory, 67% removal of total nitrogen and 96% removal of total phosphorous. The amount of total chemical oxygen demand and suspended solids was also reduced (by 74% and 82%, respectively).

- VSB Reactor: Vertical Submerged Biofilm Reactor was used to remove nitrogen and sulfide from synthetic wastewater [109]. Fixed-bed reactor made of polyvinyl chloride was able to remove 82.7% of total nitrogen and 98.2% of sulfide at third stage of the process.

- EBT Reactor: In this experiment, Chlorella sorokiniana was growing on walls of Enclosed Biofilm Tubular Reactor. In reactor made from transparent polyvinyl chloride, microalgae biofilm was used to treat piggery wastewater [111]. Algae biofilm was capable of removing carbon, ammonium, and phosphate.

- MBB Reactor: Apart from domestic wastewater, microalgae biofilms can be also used in treatment of raw water polluted by industrial activities. In 2013, Zhang tested Moving-Bed Biofilm Reactor (MBBR) for nitrogen removal, obtaining promising results [113].

- PRBC Reactor: Microalgae biofilms are helpful in removing nitrogen and phosphorous, but they can be also used in lowering the concentrations of heavy metals, such as copper, nickel or manganese. Photo-Rotating Biological Contractor was used to attach algae and microbes, which were efficiently removing heavy metals from mining wastewater [114]. From 20 to 50% of various heavy metals were taken away by the biofilm deposited on polyvinyl chloride disks partially immersed in acid mine drainage. Algae-microbial biofilm was able to withdrawn metals such as zinc, antimony, selenium, cobalt, aluminum and, as mentioned earlier, copper, nickel, and manganese. The summary of those studies is given in the table (Table 4).

4.2. Microalgae biofilms in biofuels production

There are only few studies on microalgal biofilm devoted to biofuel production [102]:

- Effect of nutrient starvation on lipid content: As it was proved by other researchers, this way of stressing algae is resulting in increase of lipid content and is so far the most common approach to increase fatty acids content in suspended cultivation The[102,116]. Unfortunately, the same effect on microalgae growing in biofilms was not observed. Lipid content was not elevated by nutrient starvation for Scenedesmus obliquus and Nitzschia palea [108]. After three days of starvation, the concentration of lipids did not changed and stayed on the level of

15% and 6% for N. palea and S. obliquus, respectively. When cultured at suspended mode, the same algal strains reached lipid level of 30% (N. palea) and 17% (S. obliquus) after three days of starvation. Nutrient starvation was not increasing the lipid content of microalgae, when grown in biofilms.

- Rotating Algal Biofilm Cultivation System: Gross constructed a Rotating Algal Biofilm cultivation system, in which he tested 16 materials as attachment surfaces [99]. The reactor was partially immersed in liquid medium. Rotations of reactor allowed the biomass that grows on substratum to alternatively enter liquid rich in nutrients and atmosphere with higher concentration of carbon dioxide. Similar approach was presented year earlier by Christenson (Fig. 5). His reactor achieved much better results regarding the biomass and fatty acid methyl esters (FAME) productivity in comparison with reactors in which microalgae were cultured at suspended mode [100]. Both studies showed that the best material for microalgae attachment is cotton. It is cheap, easy to acquire and as an attachment surface allows microalgae to achieve the highest biomass yields The[99,100].

- Attached Algal Culture System: In 2009, Johnson constructed system to grow Chlorella species intended for biofuel production with simultaneous nitrogen and phosphorous removal. Materials tested as a substrate were polystyrene foam, cardboard, polyethylene landscape fiber, loofah sponge, polyurethane foam, and nylon sponge. Among all these materials, the best in terms of biomass and total fatty acids production was polystyrene foam [115]. It was also easy to remove an algal biomass from this material and re-use polystyrene after the process (Fig. 6). The material to attach cells was placed at the bottom of moving tank. Without water movement algae tended

0 5 10 15 20 25 30

Time [days]

Fig. 5. Growth of mixed microalgae culture on different materials. Data taken from study conducted by Christenson in 2012 [100].

Fig. 6. Removing microalgae biofilm from attached cultivation system [115].

to accumulate at the bottom and created sediment rather than attach to the substratum. System was able to produce 3.2 g/m2/ day of microalgae. The lipid content was around 9%, which is much higher in comparison with maximal 5% of terrestrial crops. Dairy from wastewater was applied as a medium. Removal of total nitrogen and total phosphorous reached level of 79% and 90%, respectively [115].

4.3. Other experiments

Apart from systems to treat wastewater and produce micro-algae for biofuels, there exist researches on other applications of microalgal biofilms:

- Light/Electricity Conversion System: Biofilm can be applied to obtain energy. Biofilm-Based Light/Electricity Conversion System was developed to exchange light irradiation energy into electric current [117]. Green algae were used in the experiment, however they were working only in the presence of hetero-trophic bacteria. When the light reaches the reactor, extracellular electron transfer takes place. Electric current is generated.

- BOD removal: It is also possible to remove Biological Oxygen Demand (BOD) by application of microalgal biofilm inside Flat Plate Photobioreactor (FPP) and Tubular Packed Photobioreactor (TPP) [81]. Microalgal-bacterial biofilm is created either on beds carriers or strictly on reactor's walls. From both approaches, the second one is the most convenient, as it is not possible to achieve stability when biofilm is attached to beds carriers. In both biofilm reactors (FPP and TPP), removal rates of 92 and 108 mg BOD/L/h were achieved, in comparison with 77 mg BOD/L/h achieved in ordinary suspended reactor. It means that it is possible to conduct efficient BOD removal process with the use of microalgae and bacteria biofilm. However, the process still has certain drawbacks. Photoinhibition lowers the operation performance and the biomass accumulation during growth phase could result in reactor blockage [81].

5. Aeroterrestrial microalgae: research needs

There is a strong need for a research on aeroterrestrial micro-algae which focus on their applications in industry. Present studies, as mentioned earlier, are directed towards biofouling prevention. The potential of aeroterrestrial microalgae in biofuel production, pharmaceutics industry and other areas is not known. It is essential to investigate their properties and applications, so they can be compared with aquatic species.

Growing aeroterrestrial microalgae by non-suspended cultivation is the most intuitive step in research, than should be taken as soon as possible. This review shows that non-suspended cultivation is a promising approach in microalgae culturing, and from the natural tendency of aeroterrestrial microalgae to create biofilm it can be assumed that this kind of cultivation will be the most appropriate.

6. Conclusions

Growing microalgae in attached non-suspended systems is a novel concept. Biomass yield is comparable or higher than the same species grown at suspended mode of culturing. Consumption of water is much lower, which contributes to decreasing the costs of production. Distribution of light is improved, as it is not limited by the density of culture. Most of the cells are attached to substrate; only small part is free-floating within the medium and absorbing the light. The most significant advantage of attached

systems is no need for harvesting step. It was proven that the water content of microalgae scrapped from substratum is comparable to this of biomass after centrifugation. Avoiding this expensive and time-consuming step makes the algae production more feasible.

Application of aeroterrestrial microalgae is potentially decreasing the costs of production even more. The usage of water will be decreased to minimum, as algae will be grown in humid atmosphere, not in the medium. The light distribution is also expected to be enhanced, as no medium or floating cells would be absorbing the light. In addition, maintenance, mechanization, and scaling-up of the whole system should be easier, as huge volume of water does not obstruct operations inside the reactor.

Future work includes design of special reactor, in which humid atmosphere rich in CO2 will be created and maintained. In addition, selection of proper substrate material is important, as there are no studies on the most effective substratum to grow aeroterrestrial microalgae. To find out whether this kind of microalgae could be feasible competitor to ordinary microalgal cultivation, those investigations should be carried out in the nearest future.


The authors gratefully acknowledge the financial support for this work by the UK Engineering and Physical Sciences Research Council (EPSRC) project grant: EP/K036548/1 and FP7 Marie Curie iComFluid project grant: 312261.


[1] Wen ZY, Jiang Y, Chen F. High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development. Process Biochem 2002;37:1447-53.

[2] Mulbry W, Kondrad S, Buyer J. Treatment of dairy and swine manure effluents using freshwater algae: fatty acid content and composition of algal biomass at different manure loading rates. J Appl Phycol 2008;20:1079-85.

[3] Johnson LE. Enhanced settlement on microtopographical high points by the intertidal red alga Halosaccion glandiforme. Limnol Oceanogr 1994;39(8): 1893-902.

[4] Lehr F, Posten C. Closed photo-bioreactors as tools for biofuel production. Curr Opin Biotechnol 2009;20:280-5.

[5] Cao J, Yuan W, Pei ZJ, Davis T, Cui Y, Beltran M. A preliminary study of the effect of surface texture on algae cell attachment for a mechanical-biological energy manufacturing system. J Manuf Sci Eng 2009;131:064505.

[6] Yang J, Xu M, Zhang X, Hu Q, Sommerfeld M, Chen Y. Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance. Bioresour Technol 2011;102:159-65.

[7] Liu T, Wang J, Hu Q, Cheng P, Ji B, Liu J, Chen Y, Zhang W, Chen X, Chen L, Gao L, Ji C, Wang H. Attached cultivation technology of microalgae for efficient biomass feedstock production. Bioresour Technol 2013;127:216-22.

[8] Brennan L, Owende P. Biofuels from microalgae- a review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sust Energ Rev 2010;14:557-77.

[9] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renew Sust Energ Rev 2010;14:217-32.

[10] Doucha J, Straka F, Livansky K. Utilization of flue gas for cultivation of microalgae (Chlorella sp.) in an outdoor open thin-layer photobioreactor. J Appl Phycol 2005;17:403-12.

[11] Doucha J, Livansky K. Productivity, CO2/O2 exchange and hydraulics in outdoor open high density microalgal (Chlorella sp.) photobioreactors operated in a Middle and Southern European climate. J Appl Phycol 2006;18:811-26.

[12] Scragg AH, Illman AM, Carden A, Shales SW. Growth of microalgae with increased calorific values in a tubular bioreactor. Biomass Bioenergy 2002;23:67-73.

[13] Silaban A, Bai R, Gutierrez-Wing MT, Negulescu II, Rusch KA. Effect of organic carbon, C:N ratio and light on the growth and lipid productivity of micro-algae/cyanobacteria coculture. Eng Life Sci 2014;14:47-56.

[14] Pushparaj B, Pelosi E, Tredici MR, Pinzani E, Materassi R. An integrated culture system for outdoor production of microalgae and cyanobacteria. J Appl Phycol 1997;9:113-9.

[15] Andrade MR, Costa JAV. Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture 2007;264(1-4):130-4.

[16 [17 [18 [19 [20

[24 [25 [26

[28 [29

[32 [33

[34 [35

[36 [37 [38 [39

[40 [41 [42 [43 [44 [45 [46

Yoo C, Jun SY, Lee JY, Ahn CY, Oh HM. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresource Technol 2010;101: 571-4. [47

Krzeminska I, Pawlik-Skowronska B, Trzcinska M, Tys J. Influence of photoperiods on the growth rate and biomass productivity of green microalgae. Bioprocess Biosyst Eng 2014;37:735-41. [48

De Morais MG, Costa JAV. Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobior-eactor. J Biotechnol 2007;129(3):439-45.

Huntley M, Redalje D. CO2 mitigation and renewable oil from photosynthetic [49

microbes: a new appraisal. Mitigation Adaptation Strategies for Global

Change 2007;12(4):573-608. [50

Lopez MC, Sanchez ER, Lopez JL, Fernandez FG, Sevilla JM, Rivas J, Guerrero

mG, Grima EM. Comparative analysis of the outdoor culture of Haemato-

coccus pluvialis in tubular and bubble column photobioreactors. J Biotechnol [51


Cui Y. Fundamentals in microalgae harvesting: from flocculation to self- [52

attachment [PhD Thesis ] . Publisher: North Carolina State University; Raleigh 2013. [53

Lin YH, Leu JY, Lan CR, Lin PH, Chang FL. Kinetics of inorganic carbon utilization by microalgal biofilm in a flat plate photobioreactor. Chemo- [54

sphere 2003;53:779-87.

Cheng PF, Ji B, Gao L, Zhang W, Wang J, Liu T. The growth, lipid and [55

hydrocarbon production of Botryococcus braunii with attached cultivation. Bioresource Technol 2013;138:95-100.

Hoffmann JP. Wastewater treatment with suspended and nonsuspended [56

algae. J Phycol 1998;34:757-63.

Robinson PK, Mak AL, Trevan MD. Immobilized algae: a review. Process [57

Biochem 1986;21:122-7.

Huntley ME, Nonomura AM, Noue J. Algal culture systems. In: Huntley ME, editor. Biotreatment of agricultural wastewater. Florida: CRC Press Inc;; [58

1989. p. 111-30.

Laliberte G, Proulx D, De Pauw N, La Noue J. Algal technology in wastewater

treatment. Arch Hydrobiol Beih Ergebn Limnol 1994;42:283-302. [59

Bott TR. Industrial biofouling. B.V.: Elsevier Amsterdam; 2011.

Häubner N, Schumann R, Karsten U. Aeroterrestrial microalgae growing in

biofilms on facades- response to temperature and water stress. Microbial [60

Ecol 2006;51::258-93.

Sekar R, Nair KVK, Rao VNR, Venugopalan VP. Nutrient dynamics and [61

successional changes in a lentic freshwater biofilm. Freshwater Biol 2002;47: 1893-907.

Cowling MJ, Hodgkiess T, Parr AC, Smith MJ, Marrs SJ. An alternative [62

approach to antifouling based on analogues of natural processes. Sci Total Environ 2000;258:129-37.

Scardino AJ, Harvey E, De Nys R. Testing attachment point theory: diatom attach- [63

ment on microtextured polyimide biomimics. Biofouling 2006;22(1):55-60. Diercks S, Ch Gescher, Metfies K. Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces. J Appl Phycol 2009;21:657-68. [64

Guiry M. How many species of algae are there? J Phycol 2012;48(5):1057-63. Gaylarde CC, Gaylarde PM. A comparative study of the major microbial [65

biomass of biofilms on exteriors of buildings in Europe and Latin America. Int Biodeterior Biodegr 2005;55:131-9.

Görs S, Schumann R, Häubner N, Karsten U. Fungal and algal biomass in [66

biofilms on artificial surfaces quantified by ergosterol and chlorophyll a as biomarkers. Int Biodeterior Biodegr 2007;60:50-9. [67

Aburai N, Ohkubo S, Miyashita H, Abe K. Composition of carotenoids and identification of aerial microalgae isolated from the surface of rocks in mountainous districts of Japan. Algal Res 2013;2:237-43. [68

Eggert A, Häubner N, Klausch S, Karsten U, Schumann R. Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter. Biofouling 2006;22(1/2):79-90. [69

Elster J, Degma P, Koväcik L, Valentovä Sramkovä K, Batista Pereira A. Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia. Biologia 2008;63/ [70 6:843-51.

Gladis F, Eggert A, Karsten U, Schumann R. Prevention of biofilm growth on man-made surfaces: evaluation of antialgael activity of two biocides and [71

photocatalytic nanoparticles. Biofouling 2010;27/1:89-101. Boedeker Ch Karsten U, Leliaert F, Zuccarello GC. Molecular, biochemical and morphological data suggest an affiliation of Spongiochrysis hawaiiensis with [72 the Trentepohliales (Ulvophyceae, Chlorophyta). Phycol Res 2013;61:133-44. Hallmann Ch Stannek L, Fritzlar D, Hause-Reitner D, Friedl T, Hoppert M. [73

Molecular diversity of phototrophic biofilms on building stone. FEMS Microbiol Ecol 2013;84:355-72.

Dupuy P, Trotet G, Grossin F. Protection des monuments contre les cyano- [74

phycees en milieu abrite et humide. In: The Conservation of Stone I, R. Rossi-Manaressi (ed.), Bologna, 1976:205-21.

Ortega-Calvo JJ, Arino X, Hernandez-Marine M, Saiz-Jimenez C. Factors [75

affecting the weathering and colonization of monuments by phototrophic microorganisms. Science Total Environ 1995;167:329-41.

Morison MO, Sheath RG. Responses to desiccation stress by Klebsormidium [76 rivulare (Ulotrichales, Chlorophyta) from a Rhode Island stream. Phycologia 1985;24:129-45.

Holzinger A, Lütz C. Desiccation stress causes structural and ultrastructural [77

alterations in the aeroterrestrial green alga Klebsormidium Crenulatum

(Klebsormidiophyceae, Streptophyta) isolated from an alpine soil crust. J Phycol 2011;47:591-602.

Anandarajah K, Perumal GM, Sommerfeld M, Hu Q. Induced freezing and desiccation tolerance in the microalgae wild type Nannochloropsis sp. and Scenedesmus dimorphus. Aust J Basic Appl Sci 2011;5(1):678-86. Lukesová A, Hrouzek P, Harding K, Benson EE, Day JG. Deployment of the encapsulation/dehydration protocol to cryopreserve diverse microalgae held at the institute of soil biology, academy of sciences of the Czech Republic. Cryo-Lett 2008;29(1):21-6.

Kanavillil N, Kurissery S. Dynamics of grazing protozoa follow that of microalgae in natural biofilm communities. Hydrobiologia 2013;718:93-107. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. Bacterial biofilms in nature and disease. Annu Rev Microbiol 1987;41: 435-64.

Latour RA. Biomaterials: protein-surface interactions. Encyclopedia of biomaterials and biomedical engineering. New York: Marcel Dekker; 2004. Genzer J, Edimenko K. Recent developments in superhydrophobic surfaces and their relevance to marine fouling: a review. Biofouling 2006;22:339-60. Walker J, Surman S, Jass J. Industrial biofouling. Detection, prevention and control. John Wiley and Sons, Ltd. Hoboken; 2000.

Cooksey KE. Requirement of calcium in adhesion of a fouling diatom to glass. Appl Environ Microbial 1981;41:1378.

Pereira MO, Vieira MJ. Effects of the interactions between glutaraldehyde and the polymeric matrix on the efficacy of the biocide against Pseudomonas fluorescens biofilms. Biofouling 2001;17:93-101.

Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002;8: 881-90.

Murray RE, Cooksey KE, Priscu JC. Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia. Appl Environ Microbial 1986;52:1177-82.

Doiron K, Linossier I, Fay F, Yong J, Abd Wahid E, Hadjiev D, Bourgougnon N. Dynamic approaches of mixed species biofilm formation using modern technologies. Mar Environ Res 2012;78:40-7.

Pohlon E, Marxsen J, Küsel K. Pioneering bacterial and algal communities and potential extracellular enzyme activities of stream biofilms. FEMS Microbiol Ecol 2009;71:364-73.

Marshall KC. Microbial adhesion and aggregation. Springer-Verlag, Berlin; 1984.

Barranguet Ch Veuger B, Van Beusekom S, Marvan P, Sinke JJ, Admiraal W. Divergent composition of algal-bacterial biofilms developing under various external factors. Eur J Phycol 2005;40:1-8.

García-Meza JV, Barrangue C, Admiraal W. Biofilm formation by algae as a mechanism for surviving on mine tailing. Environ Toxicol Chem 2005;24/ 2:573-81.

Simoes M. Use of biocides and surfactants to control Pseudomonas fluorescens biofilms: role of the hydrodynamic conditions. (PhD thesis .). Tese de doutoramento em Química e Engenharia Biológica, University of Minho, Braga; 2005.

Geesey GG. Microbial exopolymers: ecological and economic considerations. ASM News 1982;48:9-14.

Matz C, Deines P, Jürgens K. Phenotypic variation in Pseudomonas sp. CM10 determines microcolony formation and survival under protozoan grazing. FEMS Microb Ecol 2002;39:57-65.

Pajdak-Stós A, Fialkowska E, Fyda J. Phormidium autumnale (Cyanobacteria) defense against three ciliate grazer species. Aq Microb Ecol 2001;23:237-44. Wingender J, Neu TR, Flemming HC. Microbial extracellular polymeric substances- characterization, structure and function. Springer, New York; 1999.

Gladis F, Schumann R. Influence of material properties and photocatalysis on phototrophic growth in multi-year roof weathering. Int Biodeterior Biodegr 2011;65:36-44.

Barberousse H, Ruot B, Yéprémian C, Boulon G. An assessment of facade coatings against colonisation by aerial algae and cyanobacteria. Build Environ 2007;42:2555-61.

Bellinzoni AM, Caneva G, Ricci S. Ecological trends in travertine colonisation by pioneer algae and plant communities. Int Biodeterior Biodegr 2003;51: 203-10.

Furey PC, Lowe RL, Johansen JR. Wet wall algal community response to infield nutrient manipulation in the Great Smoky Mountains National Park, USA. Algol Stud 2007;125:17-43.

Biggs BJF, Smith RA. Taxonomic richness of stream benthic algae: effects of flood disturbance and nutrients. Limnol Oceanogr 2002;47:1175-86. Sekar R, Venugopalan VP, Satpathy KK, Nair KVK. Rao VNR. Laboratory studies on adhesion of microalgae to hard substrates. Hydrobiologia 2004;512:109-16.

Köhler J, Hansen PD, Wahl M. Colonization patterns at the substratum-water interface: how does surface microtopography influence recruitment patterns of sessile organisms? Biofouling 1999;14(3):237-48.

Pillsbury RW, Lowe RL. The response of benthic algae to manipulation of light in four acidic lakes in northern Michigan. Hydrobiologia 1999;394: 69-81.

Chen MJ, Zhang Z, Bott TR. Effects of operating conditions on the adhesive strength of Pseudomonas fluorescens biofilms in tubes. Colloid Surface B 2005;43:61-71.

Lamb LA, Lowe RL. Effects of current velocity on the physical structuring of diatom (Bacillariophyceae) communities. Ohio J Sci 1987;87:72-8.

[78] Lazarova V, Manem J. Biofilm characterization and activity analysis in water and wastewater treatment. Wat Res 1995;29/10:2227-45.

[79] Harris CM, Kell DB. The estimation of microbial biomass. Biosensors 1985;1:17-84.

[80] Karsten U, Lembcke S, Schumann R. The effects of ultraviolet radiation on photosynthetic performance, growth and sunscreen compounds in aero-terrestrial biofilm algae isolated from building facades. Planta 2007;225: 991-1000.

[81] Munoz R, Köllner C, Guieysse B. Biofilm photobioreactors for the treatment of industrial wastewaters. J Hazard Mater 2009;161:29-34.

[82] Vieira MJ. Estudo da Formaçâo de Filmes Biológicos por Pseudomonas fluoresceins e dos efeitos associados à transferencia de massa interna e à incorporaçâo de partículas de caulino. (PhD thesis].). University of Minho, Braga; 1995.

[83] Andersen RA. Algal culturing techniques. Elsevier Academic Press, Amsterdam; 2005.

[84] Liehr SK, Wayland Eheart J, Suidan MT. A modelling study of the effect of pH on carbon limited algal biofilms. Wat. Res. 1988;22:1033-41.

[85] Lavens P, Sorgeloos P. Manual on the production and use of live food for aquaculture. Laboratory of Aquaculture and Artemia Reference Center, Rome, FAO; 1996.

[86] Starks TL, Shubert LE, Trainor FR. Ecology of soil algae: a review. Phycologia 1981;20:65-80.

[87] Nesaratnam RN. Biofilm formation and destruction on simulated heat transfer surfaces. (Ph.D. thesis].). University of Birmingham, Birmingham, UK; 1984.

[88] Silva-Aciares FR, Riquelme CE. Comparisons of the growth of six diatom species between two configurations of photobioreactors. Aquacult Eng 2008;38:26-35.

[89] Irving TE, Allen DG. Species and material considerations in the formation and development of microalgal biofilms. Appl Microbiol Biotechnol 2011;92: 283-94.

[90] Monita O, Moheimani N, Javaherdashti R, Nikraz HR, Borowitzka MA. The influence of micro algae on corrosion of steel in fly ash geopolymer concrete: a preliminary study. Adv Mater Res 2013;626:861-6.

[91] Djemai-Zoghlache Y, Isambert A, Belhaneche-Bensemra N. Electrochemical behaviour of the 316L steel type in a marine culture of microalgae (Porphyridium purpureum) under the 12/12 h photoperiod and effect of different working electrode exposure conditions on the biofilm-metal interface. J Ind Microbiol Biotechnol 2011;38:1969-78.

[92] Imase M, Watanabe K, Aoyagi H, Tanaka H. Construction of an artificial symbiotic community using a Chlorella-symbiotic association as a model. FEMS Microbiol Ecol 2008;63:273-82.

[93] Verran J, Lees G, Shakespeare AP. The effectsof surface roughness on the adhesion of Candida albicans to acrylic. Biofouling 1991;3:183-92.

[94] Percival SL, Knapp JS, Edyvean R, Wales DS. Biofilm development on stainless steel in mains water. Water Res. 1998;32:243-53.

[95] Scardino AJ, Guenther J, de Nys R. Attachment point theory revisited: the fouling reponse to a microtextured matrix. Biofouling 2008;24/1:45-53.

[96] Cooksey KE, Wigglesworth-Cooksey B. The design of antifouling surfaces: background and some approaches. Kluwer Acad Rub 1992:529-49.

[97] Briand JF, Djeridi I, Jamet D, Coupe S, Bressy C, Molmeret M, Le Berre B, Rimet F, Bouchez A, Blache Y. Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites. Biofouling 2012;28(5):453-63.

[98] Ozkan A, Berberoglu H. Cell to substratum and cell to cell interactions of microalgae. Colloids Surf B 2013;112:302-9.

[99] Gross M, Henry W, Michael C, Wen Z. Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest. Bioresour Technol 2013;150:195-201.

[100] Christenson LB, Sims RC. Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuel by-products. Biotechnol Bioeng 2012;109/7:1674-84.

[101] Schumacher JF, Aldred N, Callow ME, FinlayJA Callow JA, Clare AS, Brennan AB. Species-specific engineered antifouling topographies: correlations between the settlement of algal zoospores and barnacle cyprids. Biofouling 2007;23(5): 307-17.

[102] Hoipkemeier-Wilson L, Schumacher JF, Carman ML, Gibson AL, Feinberg AW, Callow ME, Finlay JA, Callow JA, Brennan AB. Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha). Biofouling 2004;20/1:53-63.

[103] Cooper SP, Finlay JA, Cone G, Callow ME, Callow JA, Brennan AB. Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers. Biofouling 2011;27/8: 881-91.

[104] Callow ME, Jennings AR, Brennan AB, Seegert CE, Gibson A, Wilson L, Feinberg A, Baney R, Callow JA. Microtopographic cues for settlement of zoospores of the green fouling alga Enteromorpha. Biofouling 2002;18/3: 237-45.

[105] FinlayJA, Callow ME, Ista LK, Lopez GP, Callow JA. The influence of surface wettability on the adhesion strength of settled spores of the green alga Enteromorpha and the diatom Amphora. Integr Comp Biol 2002;42:1116-22.

[106] Granhag LM, Finlay JA, Jonsson PR, Callow JA, Callow ME. Roughness-dependent removal of settled spores of the green alga Ulva (syn. Entero-morpha) exposed to hydrodynamic forces from a water jet. Biofouling 2004;20/2:117-22.

[107] Ista LK, Callow ME, FinlayJA, Coleman SE, Nolasco AC, Simons RH, Callow JA, Lopez GP. Effect of substratum surface chemistry and surface energy on attachment of marine bacteria and algal spores. Appl Environ Microbiol 2004;70/7:4151-7.

[108] Schnurr PJ, Espie GS, Allen DG. Algae biofilm growth and the potential to stimulate lipid accumulation through nutrient starvation. Bioresour Technol 2013;136:337-44.

[109] Liang Z, Xu H, Wang Y, Yang S, Du P. An investigation of a process for partial nitrification and autotrophic denitrification combined desulfurization in a single biofilm reactor. Biodegradation 2013;24/6:843-53.

[110] Chu WH, Gao N, Deng Y, Templeton MR, Yin D. Impacts of drinking water pretreatments on the formation of nitrogeneous disinfection by-products. Bioresour Technol 2011;102:11161-6.

[111] De Godos I, González C, Becares E, García-Encina PA, Munoz R. Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor. Appl Microbiol Biotechnol 2009;82: 187-94.

[112] Zamalloa C, Boon N, Verstraete W. Decentralized two-stage sewage treatment by chemical-biological flocculation combined with microalgae biofilm for nutrient immobilization in a roof installed parallel plate reactor. Bioresour Technol 2013;130:152-60.

[113] Zhang S, Wang Y, He W, Xing M, Wu M, Yang J, Gao N, Sheng G, Yin D, Liu S. Linking nitrifying biofilm characteristics and nitrification performance in moving-bed biofilm reactors for polluted raw water pretreatment. Bioresour Technol 2013;146:416-25.

[114] Orandi S, Lewis DM, Moheimani NR. Biofilm establishment and heavy metal removal capacity of an indigenous mining algal-microbial consortium in a photo-rotating biological contactor. J Ind Microbiol Biotechnol 2012;39: 1321-31.

[115] Johnson MB. Microalgal biodiesel production through a novel attached culture system and conversion parameters. (MSc thesis]). Virginia Polytechnic Institute and State University, Blacksburg; 2009.

[116] Cakmak T, Angun P, Demiray YE, Ozkan AD, Elibol Z, Tekinay T. Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii. Biotechnol Bioeng 2012;109/ 8:1947-57.

[117] Nishio K, Hashimoto K, Watanabe K. Light/electricity conversion by a self-organized photosynthetic biofilm in a single-chamber reactor. Appl Microbiol Biotechnol 2010;86:957-64.