Characteristics and potential of micro algal cultivation strategies: a review

Journal of Cleaner Production 37 (2012) 377e388

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Review

Characteristics and potential of micro algal cultivation strategies: a review Martin Koller a, e, *, Anna Salerno a, Philipp Tuffner b, Michael Koinigg b, Herbert Böchzelt b, Sigurd Schober d, Simone Pieber d, Hans Schnitzer c, Martin Mittelbach d, Gerhart Braunegg e a

Graz University of Technology, Institute of Biotechnology and Biochemical Engineering, Petersgasse 12/I, A-8010 Graz, Austria Joanneum Research Forschungsgesellschaft mbH, Elisabethstrasse 26, A-8010 Graz, Austria c Graz University of Technology, Institute of Process and Particle Engineering, Inffeldgasse 21/B, A-8010 Graz, Austria d University of Graz, Institute of Chemistry, Heinrichstrasse 28, A-8010 Graz, Austria e ARENA Arbeitsgemeinschaft für Ressourcenschonende und Nachhaltige Technologien, Inffeldgasse 23, A-8010 Graz, Austria b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 February 2012 Received in revised form 23 July 2012 Accepted 24 July 2012 Available online 3 August 2012

The review highlights the correlation between removal of various eco-toxins by micro algae and the production of high-value products thereof. It appraises established and novel strategies for micro algal cultivation, downstream processing methods for product recovery, and recent progress in algal generation of the green energy carriers biogas and biohydrogen micro algae. The suitability of selected micro algal species for various final products, and the potential of different strains for abating environmental problems are discussed. Due to the fact that low cell densities and moderate growth rates are known as the major obstacles towards a broad market penetration of micro algal products, the article shows how high cell densities and reasonable volumetric productivities can be obtained. Here, the article deals with the improvements of process design and nutrient supply regimes that are needed to achieve these goals. As demonstrated by an integrated case study, mixotrophic cultivation results in increased biomass concentration in a first cultivation step for some micro algal strains like Nannochloropsis oculata. In a second step, the fresh active algal biomass accumulates desired products via CO2 fixation, e.g. from industrial effluent gases, as the sole carbon source. This can be realized by a novel, two-stage, continuously operated closed photo-bioreactor system. After cell harvest and optimized product recovery, the value-added conversion of residual algal biomass for generation of green energy carriers, e.g. in biogas plants, constitutes another focal point of the ongoing research. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Green energy carriers Micro algae Multistage continuous production Mixotrophic cultivation mode Nannochloropsis oculata Process design Removal of eco-toxins

1. Introduction Nowadays, there exists an increasing awareness about the necessity to switch to renewable resources for generation of energy and various goods (Dovì et al., 2009). This implies the need for improving the efficiency of conversion of renewable resources by adequate catalytic processes. The application of living whole cell biocatalysts such as micro algae which are equipped with versatile metabolic abilities is a central field of research in the entire area of life sciences. The industrial scale production of green energy carriers and goods from renewables by the action of biocatalysts is generally known as “White Biotechnology”. In order to make

* Corresponding author. Graz University of Technology, Institute of Biotechnology and Biochemical Engineering, Petersgasse 12/I, A-8010 Graz, Austria. Tel.: þ43 316 873 8409; fax: þ43 316 873 8434. E-mail address: [email protected] (M. Koller). 0959-6526/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jclepro.2012.07.044

biochemical production modes of “White Biotechnology” finally competitive with common industrial approaches based on fossil resources, the improvement of several process steps is required. This encompasses an efficient upstream processing of the raw materials, the selection and improvement of the biocatalyst, the optimization of the process design and a complete utilization of side streams after downstream processing. The basic difference between “White Biotechnology” based on renewable resources and petrol-based chemical industry is illustrated in Fig. 1, where it is visible that the application of fossil feed stocks, consisting of carbon that was fixed in the bowels of earths for millions of years results in a surplus of CO2. This is in clear contrast to the principles of “White Biotechnology”; here, the released CO2 was fixed in renewable resources only a short time before. Soon, this CO2 goes “back to the fields” again by photosynthetic fixation by green plants and algae, thus closing the carbon cycle. During the past two decades, significant industrial and academic efforts were devoted globally to the development of

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Fig. 1. Scheme of the global carbon cycle. Full arrows: biotechnological production of chemicals, fuels and polymers; the mass stream for carbon is balanced. Dashed arrows: utilization of fossil feed stocks; carbon fixed in the bowels of the earth is finally converted to surplus CO2 causing a balancing problem (Malli, 2011).

process design methodologies aiming at energy conservation and waste reduction for a broad range of industrial production processes (Dunn and Bush, 2001; Fija1, 2007). The ecological impact of novel production strategies in comparison to classical modes has to be assessed already early in the stage of process development. This can be accomplished by modern tools like the Sustainable Process Index (SPI) (Krotscheck and Narodoslawsky, 1996). It is based on the assumption that a sustainable economy has its fundaments on solar energy. Solar energy can be directly used via the techniques of photovoltaics, thermal solar energy or the indirect utilization of solar energy via conversion of biomass (Gwehenberger and Narodoslawsky, 2008; Schnitzer et al., 2007). Cleaner Production is an additional method to optimize the ecobalance of a given process. It constitutes a preventive, companyspecific environmental protection initiative. It is applied as a tool to minimize waste and emissions and maximize product output. Analysing the flow of materials and energy in a company, one tries to identify options to minimize waste and emissions from industrial processes through source reduction strategies (Schnitzer and Ulgiati, 2007; Dunn and Bush, 2001). Improvements of organisation and technology help to reduce or suggest better choices in use of raw materials and energy (especially solar energy!), and to avoid the formation of various waste streams (Dovì et al., 2009; Schnitzer and Ulgiati, 2007). Micro algae can be considered as promising candidates for a broad range of applications in “White Biotechnology”; this is valid for production of goods like fine chemicals and for creation of different green energy carriers. These unicellular microbes constitute a versatile polyphyletic group of microbes with the common capability of photosynthetic fixation of CO2 for generation of various algal cell components, energy and molecular oxygen (Wang et al., 2008). To underline the significance of these powerful microbes for the eco-sphere, one should consider that the global fixation of CO2 by algae amounts to about the same quantity as the photosynthetic performance accomplished by terrestrial green plants (Velea et al., 2009). From the microbiological point of view, micro algae encompass eukaryotic and, if also including the cyanobacterial representatives (Cyanophytae; formerly also known as “blue-green algae”),

prokaryotic microbial species (Carlson et al., 2007; Koller et al., 2009). 2. Biotechnological cultivation of micro algae 2.1. General The most decisive cultivation factors determining algal growth and product formation rates are quality and quantity of nutrients (encompassing the CO2 import into the cultivation system), light supply (spectral range and photoperiod are crucial factors and have to be optimized for all micro algal species) and light intensity. Regarding the light intensity, it is of importance to avoid as well light limitation that results in so-called “dark reactions” of the cells by utilization of molecular oxygen, as photo-inhibition by excessive irradiation with photons that might even cause severe cell damage. Further, salinity (ion strength and ionic composition of the cultivation medium), pH-value, turbulence and temperature are decisive for cellular growth and product formation. Typical values found in literature report temperature ranges of 16e27  C, pHvalues of 4e11, salinities of 12e40 g L1, and light intensities of 1000e10,000 lux (reviewed by Sierra et al., 2008). During the last decades, different strategies have been developed for farming of micro algae. Comparing the cultivation set-up, one can distinguish between in- and outdoor operated systems (reviewed by Franz et al., 2012). In both cases, closed systems, so called photo-bioreactors (PBR), and open systems are available that have to ensure the sufficient supply of growing algal cells with light. This constitutes one of the most decisive factors for the apparatus design of the cultivation system. Light penetration is highly determined and often limited by the depth of the cultivation broth, cell density, the transparency of the photo-bioreactor material such as glass or plastic, and by the turbulence regime in the cultivation system (Koller et al., 2009; Coutteau, 1996). Recently, even a photobioreactor system consisting of recycled plastic water bottles was created by an Indian research group. This simple set-up was operated just by inoculating 12 plastic bottles in parallel, fixing them on a rack and connecting them to a sprinkling system with 12 ports for CO2 supply. Here, the authors report that

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promising results for lipid production by the applied algal strains can be achieved that were used for trails for biodiesel production (Topare et al., 2012). In most cases, open cultivation systems like racing ponds are cheaper to install and easy to maintain and operate (Richardson et al., 2012), but, as major drawbacks, such simple systems do not provide the possibility to efficiently control the cultivation conditions and, most of all, to prevent microbial contamination. This prevention is absolutely needed in order to operate the bioprocess under monoseptic conditions, excluding frequently occurring contamination by microbial competitors such as other eukaryotic microbes and bacteria that can endanger whole cultivation batches. This problem might be overcome by selecting specialized algal production strains that can be cultivated under extreme environmental conditions such as high salinity (described for Dunaliella), high substrate concentrations (Chlorella), high temperatures (certain thermophilic cyanobacteria like Cyanidium caldarium or Synechococcus, or the kryophilic “snow algae” Raphidonema nivale, Chloromonas pichinchae, Cylindrocystis brébissonii, and Chloromonas rubroleosa) or extreme pH-values (Spirulina) (Koller et al., 2009; Castenholz, 1969; Yamaoka et al., 1978; Hoham, 1975; Ling and Seppelt, 1993). Such extreme conditions provide the micro algal production strain with advantages during cultivation against competing microbial species. Nevertheless, it has to be emphasised that pond systems classically occupy large areas and, in many cases, it is not possible to cultivate a selected algal species with special requirements to the culture conditions in such simple systems (Mata et al., 2010). Hence, for high-productive cultivation of most micro algal species, large scale PBR systems have to be designed. 2.2. Embedding of the PHOTOCHEM research project into the scientific field of micro algal research The research project PHOTOCHEM is an ongoing cooperation by the Austrian research institutions ARENA, Graz University of Technology, University of Graz, and Joanneum Research. The project encompasses the scientific fields of microbiology, biotechnology, bioprocess engineering, analytical chemistry and chemical engineering for enhanced cultivation of selected micro algal strains. It aims at high productivities both for the phase of algal growth (formation of catalytically active biomass) and for the phase of predominant formation of intracellular products, provoked by nutritional stress conditions. An integral part of the research is to focus the cultivation of the micro algae on mixotrophic feeding strategies, using complex substrates for nutrient supply. These complex nutrients shall be derived from various industrial waste streams that can be upgraded to substrates for formation of algal biomass able to catalyze the formation of high-value products in the future. Such high-value products are for example polyunsaturated fatty acids (PUFA) like 5,8,11,14,17-eicosapentaenoic acid (EPA), docosahexaenoic (DHA), and arachidonic acid (AA) for dietary and therapeutic uses. The engineering part of the project covers the development of a two-step e continuous process using two photo-bioreactors (continuous stirred tank reactors, CSTR) in series, were active algal biomass is produced under mixotrophic conditions in the first stage, whereas the intracellular product is subsequently produced under autotrophic conditions in the second stage. Further, the separation of algal biomass from the cultivation broth using novel techniques as well as the optimized recovery of the product EPA from the producing cells constitute central tasks within the project activities. Moreover, the project team assesses potential uses of residual algal biomass that remains after product recovery for their feasibility. Especially the fermentative conversion towards the green energy carrier methane in biogas plants is one of the

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envisaged targets. Hence, it is intended to minimize waste streams and to close as many material cycles as possible. In addition, all experimental results are assessed in respect to the economic feasibility of the process and the market potential of the final products, providing the needed sets of data to create a model of the entire process. This strategy shall finally create the required knowhow enabling the design of a semi-industrial micro algae production facility on pilot scale. 2.3. Cultivation strategy e batch vs. continuous Concerning the cultivation mode, batch (discontinuous), fedbatch, repeated fed-batch, semi continuous and continuous setups are described to be applicable in biotechnological process design (Lodi et al., 2005). On a small laboratory scale, continuous strategies appear to be of special interest. This is due to the possibility of higher automation, a constant production of fresh, catalytically active cells and it enables high volumetric productivities over extended time periods. Once the equilibrium for the kinetics of growth and product formation, the so called “steady state”, is reached, biomass and products are continuously produced in constant quantity and quality. This “steady state” conditions are characterized by unvarying concentrations of substrates and products, and by constant dilution rates and residence times in the cultivation system. In contrast to dis- and semi-continuous processes, no time is needed for preparation and post-processing of the reactor system that is required prior and after each fermentation batch. As major drawback, continuous processes in biotechnology are rather complex to install and to run; hence, information on continuous biotechnological processes, especially multi-step continuous processes is still rather scarce in literature (Atli c et al., 2011). In addition, especially in the case of supplying waste streams as raw materials, the composition of the nutrient supply might to a high extent influence the growth and product formation kinetics as well as the product quality; therefore, a constant feedstock quality has to be ensured for reproducible cultivation set-ups. Furthermore, the instalment of a continuous production plant is more expensive, but e due to the higher volumetric productivities in comparison to discontinuous processes e these higher initial investment costs should be compensated within a rather short time frame. First literature reports already contain sophisticated attempts for continuous algal cultivation integrated in mollusc hatcheries, such as closed, artificially illuminated and external-loop airlift setups based on a succession of modules, each of them consisting of two transparent vertical interconnected columns (Loubière et al., 2009). Using two different micro algal strains, a single stage continuous photobioreactor process on laboratory scale was recently designed and tested. Chlorella minutissima and Dunaliella tertiolecta was investigated at different dilution rates. The authors investigated the impact of the dilution rate on biomass formation, lipid productivity and fatty acid pattern. As major outcome, the strategy turned out to be highly beneficial concerning biomass growth, but had no major effects on product formation (Tang et al., 2012). 2.4. Two-stage continuous cultivation of Nannochloropsis oculata The viability of multistageecontinuous processes for microbial formation of high-value intracellular products was recently demonstrated for polyhydroxyalkanoate (PHA) production by the prokaryotic microorganism Cupriavidus necator by the biotechnological project partners of the PHOTOCHEM project (Atli c et al., 2011). Here, a five-stage bioreactor cascade was used, producing

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Fig. 2. Chemical structure of EPA, 5,8,11,14,17-eicosapentaenoic acid (A), DHA, docosahexaenoic acid (B), and AA, arachidonic acid (C).

high densities of PHA-poor, catalytically active biomass in a first vessel under balanced nutritional conditions. This active biomass was transferred continuously into vessels 2e5, where carbon source was the only provided substrate. This provokes the enhanced accumulation of PHA by the bacterial cells due to the growth limiting nutrient conditions. Both biomass e as well as PHA-production was significantly enhanced in comparison to similar discontinuous processes in terms of volumetric and specific productivities. This is due to the fact that the characteristics of a multi-stage continuous process correspond exactly to the kinetics of biomass formation on the one hand and PHA accumulation on the other hand. Biomass growth is an autocatalytic process that, according to the chemical engineering theory, most favourably should be carried out in a stirred tank reactor. In contrast, PHA production constitutes a linear process of first order reaction that, according to the theory, should be accomplished in a tubular plug-flow reactor. Vessel 2e5 of the multistage bioreactor cascade can be regarded as a processengineering substitute for a tubular plug flow reactor (PFR). Considering the fact that micro algal growth and intracellular product accumulation obey to the same kinetic characteristics, it was manifest to use this process mode also for the cultivation of and product formation by micro algae. During the research accomplished for PHOTOCHEM, it was already possible to demonstrate the long-term stability of a twostage continuous process for micro algal cultivation. Fig. 3 shows a schematic of the two-step continuous process. Stable steady-state conditions were already maintained for several weeks. The investigated production strain, N. oculata, was cultivated continuously in a first stirred-tank photobioreactor using a mixotrophic feeding regime (see also next section), providing the cells all nutrients required for growth (proteinaceous hydrolyzate containing carbon, phosphate and nitrogen; mineral salts; CO2 for maintenance of the chloroplasts). Cell-rich cultivation broth was continuously transferred into a second stirred photobioreactor, were the only provided nutrient was CO2. Under these autotrophic conditions without availability of nitrogen source and phosphates,

the carbon flux stemming from CO2 was redirected towards predominant lipid accumulation that was the desired metabolic reaction of the strain in order to obtain high concentrations of PUFA like EPA (structure see Fig. 2). This product is of significance for dietary purposes as a food additive, and, due to its considerable market value, was selected as the target product for the PHOTOCHEM project. The continuous two-stage operation mode provided higher volumetric productivities both for the growth of catalytically active biomass and for the subsequent accumulation of EPA-rich lipids. 2.5. Cultivation conditions in dependence on the desired end products For a commercially viable production of algal biomass and its products, high cell densities are absolutely required in order to obtain reasonable volumetric productivities and to make the downstream processing economically feasible. For some algal strains, the heterotrophic cultivation, i.e. the provision of organic materials as carbon source instead of the photosynthetic fixation of CO2 (autotrophic cultivation) features a viable strategy to obtain high concentrations of catalytically active biomass. Heterotrophic feeding regimes and the combination with additional supply of CO2 (mixotrophic cultivation) are described in literature for the genera Chlorella (Bumbak et al., 2011; Martínez et al., 1997; Wu and Shi, 2007), Crypthecodinium (Bumbak et al., 2011), Galdieria (Bumbak et al., 2011), and even for cyanobacteria (Arthrospira) (Lodi et al., 2005). In this case, numerous organic waste materials can be applied as carbon substrates. The algal biomass itself is of interest as dietary or health food, as protein source for fish farming and feeding of cattle, pigs and poultry, for cosmetic purposes (especially anti-aging skin preparates; extracts from Chlorella vulgaris support collagen repair mechanisms), pharmaceutical purposes (immunity response, weight control) (Bumbak et al., 2011) and for production of biogas (Carlson et al., 2007) (see below). It must be emphasized that under such heterotrophic cultivation conditions the production of photosynthesis-based products, especially pigments, tremendously

Fig. 3. Scheme of the experimental set-up for the two-stage continuous cultivation of Nannochloropsis oculata on laboratory scale.

M. Koller et al. / Journal of Cleaner Production 37 (2012) 377e388 Table 1 Summary of micro algal pigment groups. Pigment group

Examples of the pigments Predominant Micro algal colour of the representatives algal culture

Carotinoids

Brown

Phaeophyta, Chrysophyta

Phycobillins Red Chlorophylls Green

Rhodophyta Chlorophyta

Carotenes like b-Carotin, Bixin; Xanthophylls like Violaxanthin, Astaxanthin, Lutein, Zeaxanthin Fucoxanthin Phycocyanin, Phycoerythrin Chlorophyll a (present in all photosynthetic eurcaryotic life forms)

slows down or is totally hampered. This clearly demonstrates the suitability of a continuous two-stage cultivation system, where biomass formation and product accumulation occur subsequently under different feeding regimes (heterotrophic/mixotrophic in first stage vs. autotrophic in second stage). In most phototrophic oxygen producing microbes including green micro algae, light-harvesting pigments are associated with two photosystems, generally known as PS I and PS II. In principal, three major groups of pigments are found in micro algae, being responsible for the light harvesting, CO2 fixation, and colour of the algae, as summarized in Table 1. Those pigments are considered as the algal products of highest potential for commercial success (Spolaore et al., 2006; Rao et al., 2007; Forján et al., 2007; Granado-Lorencio et al., 2009). They

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can be applied as nutrient supply due to their high contents of provitamin A (E160a), vitamin E (E306, E307, E308) (Forján et al., 2007), other pharmaceutical and medical purposes (anti-inflammatory effects, antioxidative effect, cancer prevention (Wu and Shi, 2007)), in cosmetic industry and also for food industry. In food industry, algal pigments are of high importance as food colorants (Wu and Shi, 2007). An important example is the production of astaxanthin (E161j) that is used to give salmon or trout the typical reddish color, if the farming of these fishes is supported by artificial nutrient supplements. Nowadays, the most important micro algal production strain for astaxanthin is Haematococcus pluvialis; the global market potential of this pigment was estimated with 200 million US-$ (Durmaz, 2007; Hejazi and Wijffels, 2004; Oilazola, 2005). Astaxanthin is also of importance for therapeutic applications due to its benefit in antibody production and its reported antitumour activity (Mata et al., 2010). Additionally, b-carotene is needed for the yellow coloration of egg yolk. For many dairy products such as cheese, butter or margarine, the food additive bixin (E160b) provides a yellowish to peach-colour shade. Violaxanthin features an orange colour and can technically be used as a food colorant (E161e). Fig. 4 presents the chemical structures of some of the most important algal pigments. Hence, the combination of heterotrophic and autotrophic cultivation appears to be a powerful strategy to produce high concentrations of active biomass using cheap substrates (e.g. starch hydrolysate) as first step and as second step, to switch to the production of high value compounds such as pigments or valuable

Fig. 4. Important micro algal pigments: Astaxanthin (A), Bixin (B), Violaxanthin (C), Fucoxanthin, (D); Phycocyanin (E), Phycoerythrin, (F); Chlorophyll a (G). The pigments A, B, C, and D are carotinoids, E and F are phycobillins.

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lipids like EPA as investigated in the PHOTOCHEM project by the photosynthetic fixation of CO2. In this second phase, the removal of CO2 from effluent gases by algal fixation provides the combination of mitigating surplus CO2 with value creation by the formation of marketable products. It must be emphasized that many micro algal species are able to adapt to a broad range of salinity, temperature, pH-value and nutrient conditions. The extents of production of biomass, lipids, carotenoids and carbohydrates, and the exact composition of the final products (e.g. fatty acid pattern of lipids) can vary considerably depending on the prevailing conditions in the cultivation medium (Chiu et al., 2009; Converti et al., 2009; Hodgson et al., 1991; Rao et al., 2007; Suen et al., 1987). Therefore, the applied cultivation conditions have a major impact on the productivity and quality of a desired end-product. For the selection of the appropriate production parameters, the decision has to be done from case to case to which final product the nutrient flux should be directed. 2.6. Downstream processing for cell harvest and product recovery Efficient methods for cell harvest and subsequent product recovery are pre-requisites to achieve economic viability for the entire production process. Considering the separation of micro algae from their liquid surrounding, methods of filtration, floatation, centrifugation and flocculation can be applied, depending on the micro algal species and the cell densities. For most micro algae, a two-stage combination of floatation as a tool for preconcentration of the cultivation broth, followed by a removal of the liquid by a decanter, is considered as most viable strategy. Aiming at an efficient flocculation of algal cells for further separation, this can be enhanced by adjusting the pH-value of the culture suspension, or by adding auxiliary supplies. One of the main obstacles to fully profiting of lipid-producing micro algae is the ability to successfully and efficiently extract oil from the cell biomass. After the separation of oil-rich cells from the liquid phase, the algal biomass has to be dried using methods thermo-drying or lyophilisation, both constituting rather energydemanding procedures. The dried biomass can undergo the process of product isolation. The number of well-documented procedures for extracting oil from micro algae is still scarce. Here, mechanical pressing, homogenization, milling, solvent extraction, supercritical fluid extraction, enzymatic extractions, ultrasonicassisted extraction and osmotic shock are reported in literature (Mercer and Armenta, 2011). Especially regarding solvent extraction of algal oils, the solvent throughputs have to be optimized as well as the solvent selection; here, hazardous solvents have to be avoided wherever possible. 3. Combining CO2-mitigation from effluent gases by micro algae to the production of eco-benign “green” energy carriers 3.1. Biofuels stemming from photosynthetic generation of algal lipids Caused by prevailing ecological concerns affecting our planet, the biological sequestering of CO2 by living algal cells for abatement of greenhouse gases has become a research field of increasing global significance (Carlson et al., 2007; Oonk, 2006). Using autotrophic micro algae for fixation of CO2 deriving from the exhaust gases of industrial power plants, some issues have to be considered. It is of major importance to adapt the CO2 supply to the demands of the applied production strain. Values for the optimum CO2 concentration ranges vary considerably between different algal species. Regarding the solubility of CO2 in the cultivation medium it is clear that this is influenced to a very high extend by the pH-value

that is decisive for the CO2 =HCO 3 balance. If effluent gases are introduced into algal cultures without prior processing as desired for cost effectiveness of the entire process, one has to consider that the high temperature of such exhaust gases could elevate the temperature of the cultivation system to a too high extend. This would require special technical pre-requisites for sufficient cooling capacities. In this case, thermophilic specialists among the broad variety of algal strains might also be a solution to get rid of this problem. The potential market for biodiesel by far surmounts the availability of classical vegetable oils that are not designated for other markets, e.g. for food purposes. For example, in order to meet the 20% directive in the EU from domestic production, the actual feedstock supply is not sufficient to meet the current demand. Hence, the land requirements for biofuels production would significantly surmount the theoretically available arable land for bio-energy crops (Mata et al., 2010). In addition, the efficient production of biodiesel starting from plant material is not as trivial as generally supposed; still a vast number of obstacles, especially in the up-streaming of the raw material, have to be overcome (Ponton, 2009). The combination of fixating of CO2 stemming from combustion of fossil resources by micro algae together with the conversion of this effluent gas component towards biofuels constitutes one of the most efficient strategies in abating greenhouse gases. In principle, there is no fundamental difference in the capture of CO2 from air or industrial effluent gases. The real progress in lowering the amounts of greenhouse gases is the substitution of fossil fuels by biofuels produced by the micro algae from CO2; hence, the amount of new fossil based fuel is diminished by the quantity that is substituted by the algal biofuel (Oonk, 2006). Different strains are potential candidates for micro algal biofuel production. Especially among the genera Botryococcus, Chlorella, Nannochloropsis, Neochloris, Nitzschia, Scenedesmus, Dunaliella and Schizochtyrium, several species are described to show exceptionally high amounts of lipids in their cell mass under optimal cultivation conditions (Oilazola, 2005; Oonk, 2006). For the algae, those lipids present as triacylglycerides constitute storage materials that facilitate the endurance of environmental stress conditions (Sharma et al., 2012). In the case of Botryococcus braunii, one of the best scrutinized micro algal lipid producers, a total of 75% (w/w) of hydrocarbons in cell mass were reached. The type of hydrocarbons produced by B. braunii depends on the race of this species (race A, B, L). A large number of monounsaturated and polyunsaturated and even branched hydrocarbons is produced by B. braunii; these compounds can be converted by cracking them to fuels with properties similar to those of gasoline (Rao et al., 2007). Other species produce high amounts of typical “vegetable oils” with high contents of polyunsaturated fatty acids. These oils can be classically converted to biodiesel by the well-known alkaline transesterification with alcohols like methanol (Chiu et al., 2009; Chisti, 2007; Meng et al., 2009). As the main by-product of this transesterification process, the glycerol phase can be digested anaerobically in biogas plants, can be thermally converted, or can be applied as an efficient carbon source for numerous biotechnological applications (Chisti, 2007); in addition, glycerol can be commercialized for manufacturing of e.g. cosmetic products or be applied in food industry as humectant (E422). A straightforward and plain evaluation of the outcomes of actual industrial-scale endeavours to produce biodiesel by micro algae until today leads to the conclusion that still considerable efforts have to be devoted to the optimization of this technology (Tang et al., 2012). Worldwide, a broad number of companies are claiming to be “ground breaking”, setting the standards and benchmarks in this field, and that they will be able to produce economically competitive algal biodiesel already within a very short time frame.

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In reality, most of these companies have severe shortcomings either in their technical and/or microbial know-how; in many cases, only modest quantities of biodiesel were definitely produced from algae. Especially here, the interaction of expertise in chemical engineering and microbiological comprehension is absolutely required in order to design a viable production process (Ponton, 2009). In any case, biodiesel to be used as engine fuel is not the only conceivable product stemming from algal lipids. It has to be emphasized that many algal fats contain special fatty acids with high market values, such as EPA, DHA, and AA. These fatty acids can be commercialized for pharmaceutical and therapeutic applications yielding much higher prices than after converting the lipids to biofuels for combustion. Therefore, the project PHOTOCHEM does not aim at the production of algal biofuels, but intends to enhance the production of lipids containing special fatty acids such as EPA e.g. for application as food additive. Due to the fact that also the sustainable conversion of residual algal biomass after product isolation is a topic in PHOTOCHEM, the entire process can be regarded as a “bio refinery”. Beside the CO2 supply, additional nutrients like nitrogen (as ammonia, nitrate, and organic nitrogen like urea), phosphates, sulphur as well as different minor elements have to be provided to the algae. Regarding nitrogen and sulphur, effluent gases typically contain considerable amounts of NOx and SOx mainly depending on the type of the combusted material. After being dissolved and neutralized in the aqueous cultivation medium, these gases can be converted by the algae as substrates for nitrogen and sulphur (Oonk, 2006). 3.2. Bioethanol and biogas from algal biomass After cell harvest and isolation of lipids and high-value pigments from the micro algal cells by means of extraction or mechanical disruption methods, residual biomass is generated that can be converted in different directions. Attempts have been made to generate bio ethanol from algal biomass. This can be accomplished by the fermentation of starch-rich algal biomass by the anaerobic action of yeasts (Brányiková et al., 2011). Starch and starch-like polysaccharides constitute algal reserve materials typically produced by several species among the genera of Chlorophyta, Rhodophyta, Cryptophyta, and Pyrrophyta. Due to the low yields that characterize the anaerobic ethanol production by yeasts, the largescale application of this strategy appears rather doubtful. In addition, a biotechnological production strategy using two types of micro-organisms in two separated processes (starch accumulation by micro algae followed by the anaerobic conversion of starch to ethanol) is rather complex to install and demands a rather big number of intermediary process steps. By anaerobic degradation in biogas plants, the residual biomass can be used for the generation of biogas, a more or less carbon neutral energy carrier. The generated biogas typically contains comparable amounts of the energy carrier methane and CO2. If compared to the production of hydrocarbons or biodiesel by algae, biogas generation from algal biomass is technically simple to realize. The so called “digestate” remains as residue from the biogas production. This material is rich in nutrients such as potassium, phosphates, and minor mineral components, and constitutes a precious green fertilizer in agriculture. In addition, it appears reasonable to apply the digestate as nutrient supply in subsequent algal cultivations. This recycling strategy should allow additional production of algal biomass and, in case that waste water is used as nutrient source for algal farming, act as a supplement to the nutrient supply obtained from the waste water input. Recent studies report that the potential for production of biogas is strongly dependent on the micro algal species and on the

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pre-treatment of the algal biomass. Here the application of the green alga Chlamydomonas reinhardtii is more effective in terms of biogas yield in comparison to e.g. Scenedesmus obliquus (Mussgnug et al., 2010). Recently, an integrated process for biogas production from and purification on cassava starch effluent was developed, where micro algae act as so called “bio-stabiliser agent”. The main problem of the biogas production from cassava starch effluent is the rapid decline of the pH-value due to the action of acid forming-bacteria; this antagonizes the growth of methanogen bacteria. The study demonstrates that this problem can be overcome by adding micro algae as bio-stabiliser of pH-value. At the same time, the micro algae act as purifier agent by absorbing CO2 that accrues as byproduct of the biogas production process, resulting in an increased quality of the obtained energy carrier biogas by the micro algal conditioning (Budiyono and Kusworo, 2011). The utilization of the green alga C. vulgaris SAG 211-11b for conditioning of biogas is also reported in literature (Mann et al., 2009). In this study, the biogas components CO2 and H2S could be eliminated nearby completely. Also an increase of micro algal biomass was observed, indicating the conversion of the unwanted biogas components CO2 and H2S as substrates by the micro algal cells. A life-cycle assessment (LCA) of biogas production from the micro algae C. vulgaris was performed. It highlights the main bottlenecks in this production, and compares them with the advantages and the drawbacks of mature and other immature technologies like e.g. algal biodiesel production. The authors focused on a simplified process where methane was the only recovered product; based on the results, they concluded that the optimum outcome from both the environmental and the economical point of view is a process combining lipid recovery for a fraction of the algal biomass and methane production from both raw biomass and remaining biomass after lipid extraction (Collet et al., 2011). In a recent study, the efficiency of abating CO2 by using biogas stemming from algal biomass is compared to the utilization of natural gas. It was calculated that the production of 1 ton of dry algal biomass results in avoiding 0.5 tons of CO2. It can be estimated that this value can be doubled if natural gas was replaced by coal fired energy generation, saving energy of conventional waste water treatment and replacing the energy demanding production of fertilizers by digestate (Oonk, 2006). Within the PHOTOCHEM project, the optimization of conversion of algal biomass after product isolation is a central point of research. Alternatively, residual algal biomass can be thermally converted via incineration to generate energy and ash. This can be regarded as the technologically simplest method for energy recovery from algal biomass. Similar to the digestate residues from biogas production, ash remaining from incineration can further act as a valuable agricultural fertilizer or as mineral nutrient supply for subsequent algal cultivations. 3.3. Biohydrogen production by micro algae Currently, production of the energy carrier hydrogen (H2) from renewable raw materials by the fermentative action of living cells (“bio-hydrogen”) is of increasing interest for the scientific community and provides a novel field of algal research (Chochois et al., 2009). Bio-hydrogen that can be applied in fuel cells is generally considered a future-oriented green energy carrier that is of interest for many industrial branches. Also in the case of biohydrogen production, solar energy used by the micro algae and cyanobacteria for photosynthesis is the fundamental driving force of these processes (Antal et al., 2011; Beer et al., 2009; Kapdan and Kargi, 2006). Biotechnological production of H2 is advantageous

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compared to electrolysis of water due to the rather high energy demand of this well-established technique. Under anaerobic conditions, hydrogen is produced as a by-product during conversion of a variety of organic wastes into organic acids which are subsequently used for methane generation. This so called “acidogenic phase” of anaerobic digestion of the organic wastes can be optimized in order to improve hydrogen production. Photosynthetic processes include algae which use CO2 and H2O for hydrogen gas production. Bio-hydrogen production by micro algae requires several minutes to some hours of anaerobic incubation in the dark to induce the synthesis and/or activation of enzymes involved in the hydrogen metabolism, especially a reversible hydrogenase enzyme. The hydrogenase enzyme responsible for production of H2 is highly sensitive to O2, photosynthetic production of H2 and O2 must be temporally and/or spatially separated. In a two-phase process, CO2 is first fixed into hydrogen-rich substrates such as carbohydrates during the well-investigated steps of photosynthesis (Phase 1), followed by light-mediated generation of molecular H2 when the micro algae are incubated under anaerobic conditions (Phase 2). Sulphur limitation might be a viable solution to enhance hydrogen production (Antal et al., 2011; Zhang et al., 2002). Sulphur deprivation is considered as an efficient way to trigger long-term hydrogen photoproduction in the micro-alga Chlamydomonas by a decrease in the photosystem II (water-plastoquinone oxidoreductase), which allows anaerobiosis to be reached and starch storage (Chochois et al., 2009). Recently, it has been shown that some strains of Chlorella can produce and accumulate significant volumes of hydrogen gas under anaerobic conditions and sulphur limitation such as it is reported in literature using C. reinhardtii (Antal et al., 2011; Chader et al., 2009). Nutrient deficiency severely counteracts the metabolism of the production strain. This problematic effect can be overcome by using continuous or semicontinuous feeding regimes (Antal et al., 2011). Also here, the application of the two-stage continuous process might be a suitable strategy for enhanced micro algal bio-hydrogen production. The discussed facts indicate that bio-hydrogen formation by micro algae provides a field with a huge variety of needed improvements; still the productivities for bio-hydrogen are rather low. Apart from required process engineering improvements, genetic modification of micro algae actually is regarded as the most promising strategy to efficiently generate the “green energy source” bio-hydrogen by the action of photosynthetic microbes, also in this case starting from various carbon-rich waste streams (Levin et al., 1982). 4. Removal of various toxins from water by micro algae 4.1. Removal of toxic heavy metals Several heavy metals cause severe concern because of the manifold possibilities of being exposed to them. This exposure may be environmental, occupational or residential. Small amounts of these elements are common in our environment and diet; they are actually necessary for the human metabolism, but large amounts of them may cause acute or chronic toxicity, resulting in health problems. In the case of environmental pollution by heavy metals, micro algae were identified as potential candidates to remove them from various environments, especially from industrial waste water (Khummongkol et al., 1982). Already in 1988, the green alga Chlorella fusca was described to produce a cadmium-binding complex, composed of phytochelating peptides. In addition, it was demonstrated that among the ten classes of Phycophyta, six revealed phytochelatin synthesis after exposure to Cd(II). Phytochelatin synthesis was also induced by

other heavy metal ions like Pb(II), Zn(II), Ag(I), Cu(II) and Hg(I/II). These experiments demonstrated that algae sequester heavy metals by an identical mechanism as higher plants via phytochelatins which form stable complexes with the metal cations, thus removing these pollutants from the aqueous environment (Gekeler et al., 1988). The potential of micro algae for bio-mediated removal of toxic Cr(VI) ions from aqueous solutions was demonstrated using Scenedesmus incrassatulus. Strategies to avoid Cr(VI) exposure is of special significance due to the fact that this ion is suspected to cause cancer. Among all other investigated algal species, this organism turned out to be exceptionally robust against Cr(VI) ions. The experimental set-up consisted of a split-cylinder internal-loop airlift photobioreactor that was operated continuously. A quantity of 1 mg per litre of the toxin heavily affected the pigment formation of the algae, but did not negatively influence biomass growth (Jácome-Pilco et al., 2009). Gloeothece magna, a non-toxic cyanobacterium (Cyanophyta; formerly blue-green alga) classically found in freshwater, is reported to adsorb Cd(II) and Mn(II) ions from polluted water samples. The author of the study (Mohamed, 2001) explicitly suggests that G. magna could be cultivated in water bodies contaminated by these heavy metals to decrease their toxicity. Also dry material of this cyanobacterium obtained after cell harvest and further processing, e.g. via lyophilisation or thermal drying, could be used as an efficient bio-filter system for heavy metal removal from drinking water (Mohamed, 2001). The removal of Ni(II), Fe(II/III), Hg(II), Cd(II), Cr(IV), Zn(II) or Au(II) ions from waste water by different immobilized Chlorella and Scenedesmus strains was also investigated and reported in literature (Becher, 1983; Brierley et al., 1986; Mallick, 2002; Mehta and Gaur, 2005). Here, some results indicate that immobilization of the algal cells makes them more tolerant to heavy metal ions in comparison to free cells. Hence, this strategy might imply great potential for future waste water treatment plants. In addition, this might also be a viable technological strategy for a novel bioleaching process for enrichment and recovery of e.g. gold, silver and other valuable metals (Mallick, 2002; Brierley et al., 1986). This approach appears of special significance for such metals which occur only at very low concentrations in the respective aqueous matrices, making their isolation via classical techniques economical not feasible. 4.2. Removal of formaldehyde Biodegradation of formaldehyde (methanal), a compound severely toxic for skin, eyes and the respiratory system, was demonstrated by the marine micro-alga N. oculata ST-3, a representative of the Chlorophyta. Formaldehyde is often released to marine environment via waste water from different industrial branches like paper, resin, and glue producing companies. During the reported experiments accomplished with N. oculata ST-3, formaldehyde concentration in the medium decreased in parallel with the increase of the micro algal biomass due to the potential of this micro-alga to oxidize formaldehyde to formiate. Within three weeks of cultivation, a nearby complete degradation of the toxic aldehyde in the test medium was observed. After step-wise adaptation to formaldehyde-containing cultivation media, the strain was even able to tolerate up to 20 ppm of the toxin (Yoshida et al., 2009). 4.3. Additional nutrients from waste water to be used for algal cultivation If waste water is used for nutrient supply, naturally occurring bacteria accomplish the breakdown of the organic waste materials

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to such nitrogen and phosphate sources that can subsequently easily be converted by micro algae. Hence, symbiotic interactions exist between the metabolism of the bacteria and the algae. Different waste water sources from agriculture, municipal origin or different industrial branches provide the suitable ingredients for algal nutrient supply. Additionally, the carbon present in waste water can also be converted by the algae during the heterotrophic phases of cultivation. Regarding waste water treatment plants, the disposal of activated sludge into landfills or by incineration also contributes to the formation of greenhouse gases. Nevertheless, the major effect of classical waste water treatment on the formation of greenhouse gases is due to often highly energy demanding treatment strategies, e.g. nitrogen removal via the so called “tertiary treatment” (Oonk, 2006). Especially the removal of phosphorus from waste water is a topic of globally prevailing importance (McComas and McKinley, 2008). Due to the fact that phosphorus is one of the growth decisive factors for micro algae (McComas and McKinley, 2008), and considering the fact that phosphorus nowadays is regarded to become limited in future (Steen, 1998), it is obvious that the application of such waste waters for micro algal cultivation is of high significance for environmental and biotechnological development. Again, this strategy combines the abating of ecological waste by upgrading it to a substrate for bioconversion. In order to get deeper understanding for the on-goings during the removal of nitrogen, phosphorus, and metal ions from waste water by the action of micro algae, a comprehensive study was carried out using a wide range of immobilized algal strains by Mallick (2002). A variety of algal species was used for the experiments. The study concludes that the application of micro algae has high potential for efficient waste water treatment. 4.4. Synopsis of micro algal product formation from various waste streams Fig. 5 provides a graphic schematic encompassing the potential micro algal products like lipids, pigments, biomass, molecular oxygen or bio-hydrogen that can be obtained by converting the huge variety of discussed pollution streams of different origin like waste water or industrial effluent gases. In addition, the areas of final application of the algal products, such as agriculture, energy

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generation, or pharmaceutical and nutritional purposes are indicated. The micro algal representatives discussed in this review are compared in Table 2, illustrating their major relevant products or their proved potential for abating environmentally hazardous materials. 5. Conclusion and outlook Up to date, the authors of the work at hand are not aware of any literature on the heterotrophic or mixotrophic large-scale fed-batch or even continuous cultivation of micro algae. This shortcoming in micro algal research is in contrast to exhaustive reports on lowproductive batch cultivations. By combining the application of available “liquid waste streams” like waste waters or solutions of hydrolysis products of organic rejects with the utilization of CO2 stemming from industrial effluent gases, suitable raw materials are available for both phases of the formation of high value products by selected micro algal species. Both micro algal growth and product formation have to be improved regarding the productivities. During the heterotrophic or mixotrophic phase, characterized by the formation of high densities of catalytically active algal biomass, carbon-, nitrogen- and phosphate-rich waste streams of various origins can be supplied. The second phase of the cultivation, characterized by the generation of vendible products like pigments or special lipids provides a possibility of CO2 mitigation for numerous involved industrial branches. This abating of CO2 might contribute worldwide to achieve the agreed global goals for climate protection as they are defined in the frequently discussed Kyoto Protocol to the United Nations Framework Convention on Climate Change (2005), the well-known Rio Declaration of The United Nations Conference on Environmental Protection (1992), or, more recently, at the Durban Climate Change Conference (2011) and the earth summit Rioþ20 (2012). The two-stage continuous cultivation mode provides a novel, powerful process engineering tool for high-efficient production of intracellular algal products. Closing all the material cycles in algae production, the application of those micro-organisms constitutes a powerful and sustainable strategy towards a real “White Biotechnology”. In addition, the value-added conversion of residual

Fig. 5. Combining the removal of pollutants with the production of high-value products by algal strains.

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Table 2 Discussed micro algal strains and their major marketable products, conceivable applications for production of green energy carriers, and reported potential to eliminate ecotoxins from various environments. Micro algal strain

Major products

Nannochloropsis oculata

Lipids, especially high-value PUFAs pigments Biodiesel by transesterification; Removal of formaldehyde Biogas from water Biogas e Protein: fish farming, dietary or health food Lipids, especially high-value fatty acids (linoleic acid and g-linolenic acid) Pigments (phycocyanin, carotenoids) e Protein: dietary or health food; fish farming Biogas and feeding of cattle, pigs and poultry and cosmetic purposes Protein: fish farming and feeding of cattle, Biogas Removal of various heavy pigs and poultry metals from water

Arthrospira sp. (Spirulina sp.)a

Chlorella vulgaris

Chlorella sp.

Green energy carrier

Crypthecodinium cohnii Scenedesmus sp.

Lipids, especially high-value PUFAs Lipids

Biogas Biogas

Haematococcus pluvialis

Pigments (astaxanthin)

Biogas

Dunaliella salina Botryococcus braunii

Pigments (b-carotene; bixin, zeaxanthin) Hydrocarbons; Pigments (violaxanthin, lutein) Lipids Lipids Lipids Lipids Starch and starch-like polysaccharides

Biogas Gasoline-like biofuel from cracking of hydrocarbons Biodiesel by transesterification Biodiesel by transesterification Biodiesel by transesterification Biodiesel by transesterification Bioethanol by fermentation of polysaccharides by yeasts

Schizochtyrium sp. Nitzschia sp. Neochloris oleoabundans Dunaliella primolecta Representatives of Chlorophyta, Rhodophyta, Cryptophyta, and Pyrrophyta. Chlamydomonas reinhardtii, e Scenedesmus obliquus Chlamydomonas sp. e

Removal of eco-toxin

Reference Chiu et al., 2009, Yoshida et al., 2009 Mendes et al., 2006; Chaiklahana et al., 2008

Bumbak et al., 2011; Mann et al., 2009; Collet et al., 2011 Becher, 1983, Brierley et al., 1986, Mallick, 2002, Mehta and Gaur, 2005 e de Swaaf et al., 2003 Removal of various heavy Becher, 1983, Brierley metals from water et al., 1986, Mallick, 2002, Mehta and Gaur, 2005 e Durmaz, 2007; Hejazi and Wijffels, 2004; Oilazola, 2005 e Granado-Lorencio et al., 2009 e Rao et al., 2007 e e e e e

Chiu et al., 2009 Chiu et al., 2009 Chisti, 2007 Chisti, 2007 Brányiková et al., 2011

Biogas

e

Mussgnug et al., 2010

Biohydrogen; Biogas

e

Binding of various heavy metals from water Removal of Cr(VI) ions from water Removal of Cd(II) and Mn(II) ions from water

Antal et al., 2011, Chader et al., 2009, Chochois et al., 2009 Gekeler et al., 1988

Chlorella fusca

e

Biogas

Scenedesmus incrassatulus

e

Biogas

Gloeothece magnaa

e

Biogas

Jácome-Pilco et al., 2009 Mohamed, 2001

PUFAs Polyunsaturated fatty acids. a Cyanobacteria.

algal biomass that remains after product isolation for generation of the energy carrier biogas gives novel impulses for the area of “green energy”. This goes in parallel to the progress in bio-hydrogen production by micro algae. This novel cultivation set-up could also be applied to novel micro algal strains improved by genetic engineering approaches. Fundamental knowledge enabling tailor-made strain design may be derived from advanced metabolic flux analyses. Considerable progress in this direction is very likely within a rather short time frame due to the successful research accomplished during the past 10e20 years, which resulted in the complete sequencing of the first micro algal genomes (Wu and Shi, 2007). In addition, the continuous formation of biomass under balanced nutritional conditions in a first stage followed by increased, continuous product formation provoked by nutritional stress in a second stage could also be applied to other microbial “cell factories”. Here, the accumulation of oils by oleaginous yeasts, provoked by nitrogen limitation in the second stage, appears worthwhile to be investigated. These oils obtained from yeasts might be used as a novel raw material for biodiesel production. In future, genetic engineering and technological optimization of production facilities might open the route for the efficient micro algal production of bio-hydrogen as an additional sustainable energy carrier. Further, the removal of various pollutants in

typically aqueous environments, such as eco-toxins like heavy metals is a seminal field for application of micro algae in the coming years. The comprehensive implementation of already highly advanced techniques of photovoltaic for generation of electrical power and solar thermal energy needed for running the cultivation system und the downstream processing can provide a sustainable strategy for an autarkic energy supply of the entire algal-based production plant. Following this strategy, one takes direct profit from solar energy firstly for the photosynthetic fixation of CO2 by the algal cells, and, secondly, for energy and heat generation to run the production facilities. Uniting the possible enhancements of each process step, one can definitely make substantial progress towards a cost-efficient algalbased technology. In any case, the development of really efficient processes for manufacturing of algal products starting from diverse waste streams needs the narrow cooperation of experts from different scientific fields. Chemical engineers, microbiologists, genetic engineers and experts in the fields of Life Cycle Assessment and Cleaner Production Studies have to concentrate their special expertise and know-how in order to close the existing gaps between promising data from the laboratory scale to industrial realization. It has to be emphasize that a direct scale up from laboratory to industrial implementation is not realistic. Therefore,

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investigations on pilot scale are required in order to critically and plain assess the outcomes for different laboratories. Until today, especially the lacking interaction of skills in process design and well-grounded understanding of intracellular on-goings hampers the broad industrial implementation of these powerful phototrophic cell-factories. Acknowledgement The research embedded in this review is enabled by the project “PHOTOCHEM e Mikroalgen zur Herstellung von Chemikalien e Grundlagen der Herstellung und Aufarbeitungstechnologien” (“PHOTOCHEM e micro algae for production of chemicals e basics of production and technologies for product isolation”; project number 5036). The authors are gratefully acknowledging the financial support provided by the Austrian Province of Styria (Land Steiermark) from the budget of the “Zukunftsfonds Steiermark”. References Antal, T.K., Krendeleva, T.E., Rubin, A.B., 2011. Acclimation of green algae to sulfur deficiency: underlying mechanisms and application for hydrogen production. Appl. Microbiol. Biotechnol. 89, 3e15. Atli c, A., Koller, M., Scherzer, D., Kutschera, C., Grillo Fernandes, E., Horvat, P., Chiellini, E., Braunegg, G., 2011. Continuous production of poly([R]-3hydroxybutyrate) by Cupriavidus necator in a multistage bioreactor cascade. Appl. Microbiol. Biotechnol. 91, 295e304. Becher, E.W., 1983. Limitations of heavy metal removal from waste water by means of algae. Water Res. 17 (4), 459e466. Beer, L.L., Boyd, E.S., Peters, J.W., Posewitz, M.C., 2009. Engineering algae for biohydrogen and biofuel production. Curr. Opin. Biotechnol. 20 (3), 264e271. Brányiková, I., Marsálková, B., Doucha, J., Brányik, T., Bisová, K., Zachleder, V., Vítová, M., 2011. Microalgae e novel highly efficient starch producers. Biotechnol. Bioeng. 108 (4), 766e776. Brierley, J.A., Brierley, C.L., Goyak, G.M., 1986. AMT-BIOCLAIM: a new wastewater treatment and metal recovery technology. In: Lawrences, R.W. (Ed.), Proc. of the 6th Int. Symp. Biohydrometallurgy, pp. 291e304. Budiyono, B., Kusworo, T.D., 2011. Biogas production from cassava starch effluent using microalgae as biostabilisator. Int. J. Sci. Eng. 2 (1), 4e8. Bumbak, F., Cook, S., Zachleder, V., Hauser, S., Kovar, K., 2011. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Appl. Microbiol. Biotechnol. 91, 31e46. Carlson, A.S., van Beilen, J.B., Moeller, R., Clayton, D., 2007. Micro- and Macroalgae: Utility for Industrial Applications. CPL Press, Tall Gables, The Sydings, Speen, Newbury, Berks RG14 1RZ, UK. Castenholz, R.W., 1969. Thermophilic blue-green algae and the thermal environment. Bacteriol. Rev. 33 (4), 476e504. Chader, S., Hacene, H., Agathos, S.N., 2009. Study of hydrogen production by three strains of Chlorella isolated from the soil in the Algerian Sahara. Int. J. Hydrogen Energy 34, 4941e4946. Chaiklahana, R., Chirasuwana, N., Lohab, V., Bunnag, B., 2008. Lipid and fatty acids extraction from the cyanobacterium Spirulina. Sci. Asia 34, 299e305. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology 25, 294e306. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833e838. Chochois, V., Dauvilleé, D., Beyly, A., Tolleter, D., Cuiné, S., Timpano, H., Ball, S., Cournac, L., Peltier, G., 2009. Hydrogen production in Chlamydomonas: photosystem II-dependent and independent pathways differ in their requirement for starch metabolism. Plant Physiol. 151, 631e640. Collet, P., Hélias, A., Lardon, L., Ras, M., Goy, R.A., Steyer, J.P., 2011. Life-cycle assessment of microalgae culture coupled to biogas production. Bioresour. Technol. 102 (1), 207e214. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process. 48 (6), 1146e1151. Coutteau, P., 1996. Micro algae. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Life Food for Aquaculture. FAO, Rome. FAO Fisheries Technical Paper. No. 361. de Swaaf, M.E., Sijtsma, L., Pronk, J.T., 2003. High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol. Bioeng. 81 (6), 666e672. Dovì, V.G., Friedler, F., Huisingh, D., Klemes, J.J., 2009. Cleaner energy for sustainable future. J. Cleaner Prod. 17 (10), 889e895. Dunn, R.F., Bush, G.E., 2001. Using process integration technology for cleaner production. J. Cleaner Prod. 9, 1e23.

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