Biodesalination: A Case Study for Applications of...Biodesalination: A Case Study for Applications...

Update on Usage of Cyanobacteria for Water Treatment

Biodesalination: A Case Study for Applications ofPhotosynthetic Bacteria in Water Treatment1[C]

Jaime M. Amezaga, Anna Amtmann*, Catherine A. Biggs, Tom Bond, Catherine J. Gandy,Annegret Honsbein, Esther Karunakaran, Linda Lawton, Mary Ann Madsen, Konstantinos Minas,and Michael R. Templeton

School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne NE1 7RU,United Kingdom (J.M.A., C.J.G.); Institute of Molecular, Cell and Systems Biology, College of Medical,Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom (A.A., A.H.,M.A.M.); Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD,United Kingdom (C.A.B., E.K.); Department of Civil and Environmental Engineering, Imperial College London,London SW7 2AZ, United Kingdom (T.B., M.R.T.); and Institute for Innovation, Design and Sustainability,Robert Gordon University, Aberdeen AB10 7AQ, United Kingdom (L.L., K.M.)

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the nextdecades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in theoceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization ofdesalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for thispurpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-saltreservoir within the saline water. The latter could be used as an ion exchanger throughmanipulation of transport proteins in the cellmembrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for theexploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, geneticmanipulation, ion transport, cell-water separation, process design, safety, and public acceptance.

Bacteria are commonly employed for the purificationof municipal and industrial wastewater but until now,established water treatment technologies have not takenadvantage of photosynthetic bacteria (i.e. cyanobacteria).The ability of cyanobacterial cultures to grow at highcell densities with minimal nutritional requirements (e.g.sunlight, carbon dioxide, and minerals) opens up manyfuture avenues for sustainable water treatment applications.

Water security is an urgent global issue, especiallybecause many regions of the world are experiencing,or are predicted to experience, water shortage con-ditions: More than one in six people globally arewater stressed, in that they do not have access to safedrinking water (United Nations, 2006). Ninety-sevenpercent of the Earth’s water is in the oceans; conse-quently, there are many efforts to develop efficientmethods for converting saltwater into freshwater.Various processes using synthetic membranes, suchas reverse osmosis, are successfully used for large-scale

desalination. However, the high energy consumption ofthese technologies has limited their application predomi-nantly to countries with both relatively limited freshwaterresources and high availability of energy, for example, inthe form of oil reserves.

The development of an innovative, low-energy bio-logical desalination process, using biological membranesof cyanobacteria, would thus be both attractive and per-tinent. The core of the proposed biodesalination process(Fig. 1) is a low-salt biological reservoir within seawaterthat can serve as an ion exchanger. Its development canbe separated into several complementary steps. The firststep comprises the selection of a cyanobacterial strain thatcan be grown to high cell densities in seawater withminimal requirement for energy sources other than thosethat are naturally available. The environmental condi-tions during growth can be manipulated to enhancenatural extrusion of sodium (Na+) by cyanobacteria. Inthe second step, cyanobacterial ion transport mechanismsmust be manipulated to generate cells in which sodiumexport is replaced with intracellular sodium accumula-tion. This will involve inhibition of endogenous Na+ ex-port and expression of synthetic molecular units thatfacilitate light-driven sodium flux into the cells. A robustcontrol system built from biological switches will be re-quired to achieve precisely timed expression of the salt-accumulating molecular units. The third step consistsof engineering efficient separation of the cyanobacterialcells from the desalinated water, using knowledge ofphysicochemical properties of the cell surface and their

1 This work was supported by the Engineering and Physical Sci-ences Research Council.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Anna Amtmann ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.113.233973

Plant Physiology�, April 2014, Vol. 164, pp. 1661–1676, www.plantphysiol.org � 2014 American Society of Plant Biologists. All Rights Reserved. 1661

https://plantphysiol.orgDownloaded on January 13, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.113.233973

https://plantphysiol.org

natural ability to produce extracellular polymeric sub-stances (EPSs), which aid cell separation while preservingcell integrity. The fourth step integrates the first threesteps into a manageable and scalable engineering process.The fifth and final step assesses potential risks and publicacceptance issues linked to the new technology.

In this review, we outline the state of knowledge andavailable technology for each of the steps, as well assummarize the current knowledge gaps and technicallimitations in employing a large-scale water treatmentprocess using cyanobacteria. Before discussing these is-sues, we provide some background information on theusage of cyanobacteria in biotechnology and the impactof sodium on cellular functions of cyanobacteria. Theexample of biodesalination provides a good vehicle todiscuss the suitability of photosynthetic bacteria for watertreatment more generally. The issues addressed in thisreview are relevant for a wide range of biotechnologicalapplications of cyanobacteria, including bioremediationand biodegradation as well as the generation of biofuels,natural medicines, or cosmetics.

CYANOBACTERIA IN BIOTECHNOLOGY

Cyanobacteria are a phylum of photosynthetic,oxygen-producing bacteria, with a long evolutionary

history (Altermann and Kazmierczak, 2003). Because ofthe process of complementary chromatic adaptation(Bennett and Bogorad, 1973), cyanobacteria can utilize awide spectrum of photosynthetically active radiation astheir primary source of energy. Their long evolutionaryhistory has allowed them to adapt to a wide range ofenvironmental conditions and to occupy a vast array ofecological niches. Modest growth requirements combinedwith high adaptability generate a potential for harmfulalgal blooms, which have earned these organisms somebad publicity. However, cyanobacteria have contributedto human nutrition for millennia either directly as a foodsource or indirectly through nitrogen fixation in ricepaddies (Thajussin and Subramanian, 2005; Landsberg,2010). More recently, biotechnological applications ofcyanobacteria have allowed for their utilization as animalfeeds and human food supplements and as producers ofbioenergy, cosmetics, and anticancer and anti-HIV drugs(Spolaore et al., 2006).

In the context of environmental cleanup, Oscillatoriasalina, Plectonema terebrans, and Aphanocapsa sp. havebeen used successfully for the degradation of crude oilin seawater (Raghukumar et al., 2001). The applicabilityof cyanobacteria extends to the remediation of heavymetals (e.g. cadmium by Tolypothric tenuis) or even thereclamation of precious metals (e.g. gold by Plectonema

Figure 1. Proposed usage of cyanobacterial cultures for water treatment. A, Hypothetical water treatment station. Situated in basinsnext to the water source, sun-powered cell cultures remove unwanted elements from the water. The clean water is separated from thecells for human uses. The produced biomass is available for other industries. The proposed biodesalination process is based on thefollowing steps. B, Photoautotrophic cells divide to generate high-density cultures. C, The combined cell volume is low in salt as a resultof transport proteins in the cell membrane that export sodium using photosynthetically generated energy. D, Through environmental andgenetic manipulation, salt export is inhibited and replaced with transport modules that accumulate salt inside the cells. This process isagain fueled by light energy. E, Manipulation of cell surface properties separates the salt-enriched cells from the desalinated water.

1662 Plant Physiol. Vol. 164, 2014

Amezaga et al.



boryanum; Inthorn et al., 1996; Lengke et al., 2006). Anindication for possible usage of cyanobacteria for desali-nation was the reclamation of saline soils in India and theSoviet Union using endemic strains (Apte and Thomas,1997; Singh and Dhar, 2010). Thus, removal of cyano-bacterial mats formed after rainfall also removed saltfrom the soil. Further investigation of Anabaena torulosa(a brackish strain) and Anabaena sp. strain L-31 (a fresh-water strain) demonstrated that 90% of the salt accumu-lated was bound to EPSs at the cell surface, whereas theremainder was internalized and osmotically active. Thefreshwater strain showed a higher net sodium uptakethan the brackish strain, probably because of the highersodium efflux capacity of the latter. Interestingly, the in-flux of sodiumwas diminished in both strains by alkalinepH, the high amount of extracellular potassium, or thepresence of nitrates or ammonium (Apte and Thomas,1983; Apte et al., 1987). These observations suggested thatenvironmental triggers could be used to alter the mag-nitudes of sodium influx and efflux through endogenoustransport systems.

SELECTION OF SUITABLECYANOBACTERIAL STRAINS

Strain selection for biotechnological applications needsto be guided by the purpose and the environment of theenvisaged process. With respect to biodesalination, can-didate strains should meet a few key criteria. The cultureshould be fast-growing to allow for the generation ofhigh cell density within a short time, thereby generatinga large cumulative internal volume and a large total cellsurface. The strain should be able to grow over a widerange of external salt concentrations; the cells should beable to adjust osmotically and to effectively export Na+

during growth. To allow for cell separation from thewater and other posttreatment procedures, the cellsshould preferably be unicellular, possess a cell wall andEPS, and have the capacity to adjust their buoyancy(e.g. through intracellular gas vesicles). Finally, to fa-cilitate genetic manipulation, the cells should be ame-nable to transformation techniques and their genomesequence should be known.Based on these criteria and an initial screen carried out

in one of our laboratories (Fig. 2), two strains emerge asattractive candidates for biodesalination: the freshwatereuryhaline Synechocystis sp. strain PCC 6803 (Richardsonet al., 1983) and the marine-euryhaline Synechococcus sp.strain PCC 7002 (formallyAgmenellum quadruplicatum PR-6;Ludwig and Bryant, 2012). Both strains are unicellular,are capable of axenic growth, and are easy to maintainunder laboratory conditions. The genomes of both orga-nisms have been sequenced (Kazusa DNA Research In-stitute, 2013) and successful transformation with foreignDNA has been reported (see below). A particular ad-vantage of Synechococcus sp. strain PCC 7002 is its highgrowth rate. Generation times of less than 3 h have beenreported, making this strain the fastest dividing cyano-bacterium and one of the fastest growing photosynthetic

organisms (Van Baalen et al., 1971). Both strains havebeen used extensively as models for the study of photo-synthesis. This research has already provided a wealth ofscientific knowledge, including information on physio-logical adaptations to salinity and other environmentalfactors (Nakamura et al., 2000; Ludwig and Bryant, 2012).

MANIPULATION OF ENDOGENOUS SODIUMTRANSPORT IN CYANOBACTERIA

Any usage of unicellular systems such as cyanobac-teria for the removal of sodium (Na+) from seawater orbrackish water requires an understanding of the poten-tial effects of Na+ on cellular functions, which in turndepend on the Na+ concentration. SomeNa+ is necessaryfor nutrient uptake (e.g. Na+-dependent HCO3

2 trans-port), nitrate assimilation, nitrogen fixation, and photo-synthesis (Apte and Thomas, 1983; Maeso et al., 1987;Espie et al., 1988). Na+ is also required for cell division inheterotrophic cyanobacteria and for pH homeostasis inalkaline environments (Miller et al., 1984). Deleteriouseffects become apparent when the intracellular sodiumconcentration exceeds a certain level, including desta-bilization of the fatty acids in the cell membrane (Huflejtet al., 1990), inhibition of electron transport betweenH2O and PSII (Allakhverdiev and Murata, 2008), and acomplete halt of photoautotrophic growth (Bhargavaet al., 2003). The exact level at which Na+ becomes toxicdepends on both endogenous and environmental factorsand differs between strains.

Successful salt acclimation of cyanobacterial cells de-pends on ambient concentrations and length of the ex-posure (Marin et al., 2004; Hagemann, 2010). It is amultistage process that includes the readjustment ofionic and osmotic potentials as well as wider physio-logical changes. Turgor adjustment is one of the earliest

Figure 2. Prescreening of cyanobacterial cultures for strain selection.The effects of different media and environmental conditions on the per-formance of cyanobacterial cultures can be tested under controlled con-ditions in the laboratory. [See online article for color version of this figure.]

Plant Physiol. Vol. 164, 2014 1663

Cyanobacteria for Biodesalination



responses to salt stress (Blumwald et al., 1983). It involvesthe biogenesis and accumulation of compatible solutessuch as glycosylglycerol and Suc (Porchia and Salerno,1996; Engelbrecht et al., 1999). Moreover, increasedosmolyte uptake has been observed in some strains undersalt stress, and this uptake appeared to alleviate some ofthe effects caused by salinity (Fulda et al., 1999). If saltstress persists, ionic adjustment becomes increasingly im-portant, in particular the active extrusion of Na+ throughNa+/H+ antiporters, as well as P-type Na+-ATPases (Marinet al., 2004; Wiangnon et al., 2007).

Environmental manipulations can make use of factorsthat directly or indirectly alter the metabolism of the or-ganism. The primary metabolism of cyanobacteria islargely based on photosynthesis and is hence stronglyregulated by light. By altering the photoperiod, light in-tensity, or wavelength, metabolic processes can be in-duced or inhibited literally by the flick of a switch. Theavailability of carbon, nitrate, and phosphate also exertssignificant control over growth, metabolism, and energystatus. In particular, cotransport of bicarbonate, phos-phate, and nitrate with Na+ (Shibata et al., 2002; Matsudaet al., 2004; Baebprasert et al., 2011) opens opportunitiesto use these macronutrients to modulate Na+ uptakerates. Altering the cell’s energy status through metal de-ficiencies will affect active Na+ export from the cell, whichconsumes a large proportion of the cell’s ATP. Magne-sium in chlorophyll and iron in heme groups are essentialcomponents of the photosystem and are hence requiredfor photosynthetic activity, whereas inorganic phosphateis required for oxidative phosphorylation. Deficiency ofthese elements is the most common reason for culturesentering the stationary phase, and it can thus be expectedthat cells lose their capacity to exclude Na+ toward theend of the growth period. Furthermore, metabolic activityis affected by changes in pH and temperature. A sys-tematic assessment of the effects of individual factors, andof their combinations, on Na+ transport in Synechococcussp. strain PCC 7002 and Synechocystis sp. strain PCC 6803is now required to provide a set of environmental triggersthat can be used to alter Na+ exchange between the cellsand the surrounding water.

GENETIC MANIPULATION OF CYANOBACTERIA

The two methods that are most commonly used fortransferring foreign genetic material into cyanobacteriaare natural transformation and conjugation. Several de-tailed reviews have been published on the genetic ma-nipulation of cyanobacteria in general (Koksharova andWolk, 2002; Vioque, 2007; Heidorn et al., 2011; Wilde andDienst, 2011). Here we will only give a short overviewwith emphasis on available tools for Synechococcus sp.strain PCC 7002 and Synechocystis sp. strain PCC 6803.

Natural Transformation

Natural transformation involves the spontaneousuptake of DNA from the environment and subsequentintegration into the host genome. Both Synechococcus

sp. strain PCC 7002 (Stevens and Porter, 1980; Essichet al., 1990; Frigaard et al., 2004) and Synechocystis sp.strain PCC 6803 (Grigorieva and Shestakov, 1982; Bartenand Lill, 1995; Heidorn et al., 2011) are naturally trans-formable, although the process of DNA uptake is incom-pletely understood. Among other factors, type IV pili,which are also responsible for cell mobility, are an im-portant part of the natural competence of Synechocystis sp.strain PCC 6803 (Yoshihara et al., 2001, 2002). Onlydouble-stranded DNA can be used for natural transfor-mation, but it is converted into single-stranded DNA as itpasses through the cell envelope. Inside the cell, thedouble-stranded state is restored during recombinationwith the chromosomal or plasmid DNA of the host (Essichet al., 1990; Barten and Lill, 1995). A calcium-dependentnuclease, located in or on the plasma membrane, wasproposed to be responsible for the degradation of oneof the two strands during DNA uptake in Synechocystis sp.strain PCC 6803 (Barten and Lill, 1995). In Synechocystis sp.strain PCC 6803, no further fragmentation of extracellularDNA incorporated into the cell in this manner was ob-served (Barten and Lill, 1995; Kufryk et al., 2002).

So-called integrative or suicide plasmids are used fornatural transformation in the laboratory. These plasmidsare able to replicate in Escherichia coli, which is used forcloning of the gene before transfer to the cyanobacterialhost. They allow the researcher to position the gene ofinterest between two flanking regions of DNA that arehomologous to sequences of the cyanobacterial genome,the so-called neutral sites. Neutral sites are regionswhose deletion or interruption has produced no pheno-typic effect under all growth conditions investigated thusfar. Neutral sites are generally found in silent or redun-dant genes as opposed to intergenic or 39-untranslatedregions, which can execute regulatory functions on geneexpression (Wilde and Dienst, 2011). In Synechocystis sp.strain PCC 6803 and Synechococcus sp. strain PCC 7002,integration of foreign DNA between the two flankinghomologous regions usually occurs by a double cross-over event mediated by the highly efficient homologousrecombination system of these strains. The recombina-tion efficiency depends on the length of the homologousstretches. The optimal length is different for differentstrains, but generally the longer the better (Labarre et al.,1989; Heidorn et al., 2011; Xu et al., 2011). Covalentlyclosed or linearized plasmids as well as PCR products ofthe region of interest can be used in natural transfor-mation. For Synechocystis sp. strain PCC 6803, transfor-mation with circular plasmid DNA was found to beapproximately 30% more efficient than transformationwith linearized plasmid DNA (Kufryk et al., 2002). ForSynechococcus sp. strain PCC 7002, the use of linearfragments was recommended to achieve high transfor-mation efficiency (Xu et al., 2011).

Conjugation

DNA transfer by conjugation consists of plasmidexchange between different bacteria. In the laboratory,three strains are typically used for conjugation, also


Amezaga et al.



called triparental mating: the host cyanobacterium andtwo E. coli strains. One E. coli strain carries the vectorcontaining the gene of interest (cargo plasmid) and thesecond E. coli strain carries the conjugal plasmid. Ifadditional helper plasmids are needed, they usuallyjoin the cargo plasmid in the first E. coli strain. Mixingof the two E. coli strains causes the conjugative plasmidto transfer to the E. coli strain that carries the cargo andhelper plasmids. The latter is then competent to con-jugate with the subsequently added cyanobacterialstrain and to transfer the cargo plasmid to the newhost (Vioque, 2007; Wilde and Dienst, 2011).The cargo plasmids used for conjugation are vectors

capable of autonomous replication in the host cyanobac-terium, as well as in E .coli, where the initial cloning takesplaces. Two types of vectors can be distinguished. Shuttlevectors are hybrids between a native cyanobacterialplasmid and an E. coli plasmid and therefore carry twodifferent origins of replication, one that is specific for theparticular cyanobacterium and one that is specific forE. coli. Broad host range vectors carry only one replicon,which functions in many different bacterial hosts, in-cluding cyanobacteria and E. coli (Heidorn et al., 2011).For conjugal transfer of both types of vectors, certain

additional genetic elements are essential. Most impor-tantly, in the donor cell, a relaxase/nickase of the mo-bility gene family (mob genes) recognizes and cleaves aspecific site within an origin of transfer. The DNA strandwith the covalently bound relaxase protein is displacedfrom the plasmid by an ongoing conjugative DNA rep-lication process. Through interaction of the relaxase withcomponents of a multiprotein, membrane-associatedmating pair formation complex, a type IV secretion sys-tem (tra genes), it is transported to the recipient cell to-gether with the attached DNA. In the recipient cell, therelaxase catalyzes the ligation of the transported DNA toreconstitute the conjugated plasmid (Smillie et al., 2010).The origin of transfer is the only sequence required in cisfor a plasmid to be conjugally transmissible, which iswhy both the shuttle vector and the broad host vectorscarry this DNA sequence.

Other Techniques for DNA Transfer

Protocols enabling DNA transfer through electropora-tion have been developed for Synechocystis sp. strain PCC6803 (Marraccini et al., 1993; Zang et al., 2007), but cellrecovery after the procedure is slow and there are reportsthat this technique increases mutation rates in some cy-anobacteria (Bruns et al., 1989; Muhlenhoff and Chauvat,1996). In the future, transfer of DNA through cyano-bacterial viruses (cyanophages) could become an attrac-tive alternative, although appropriate genetic tools fortransduction have not yet been published. However,nonlytic cyanophages that infect marine Synechococcus sp.and have their genome stably maintained within the hosthave already been described (McDaniel et al., 2002).Furthermore, it is known that some cyanophages have abroad host range and can cross infect both Prochlorococcus

and closely related Synechococcus sp., which has beenimplicated in horizontal gene transfer of photosynthesis-related genes (Sullivan et al., 2003; Weigele et al., 2007).Those types of phages have potential for the develop-ment of genetic tools.

Technique of Choice and Current Limitations

The technique of choice for the genetic manipulation ofSynechocystis sp. strain PCC 6803 and Synechococcus sp.strain PCC 7002 will depend on how the foreign geneinformation should be maintained in the host cyano-bacterium. As mentioned above, the process of naturaltransformation involves DNA linearization and conversionto a single strand (Porter, 1986), which makes this tech-nique unsuitable for genes on an autonomously replicatingplasmid. In this case, conjugation is the method of choicebecause it ensures that a circular plasmid resides in thehost at the end of the transfer (Vioque, 2007). Integrationinto the host genome by natural transformation is desir-able when long-term inheritance is the goal. It also po-tentially reduces gene dose variation caused by copynumber variations of autonomously replicating plasmids.The downside of incorporation of foreign DNA by ho-mologous recombination into the genome is that cyano-bacteria generally have multiple copies of the chromosome(e.g. 12 in Synechocystis sp. strain PCC 6803), and hetero-zygous cells are thus created. Subsequent segregation overseveral generations is needed to ensure that the foreignDNA is present in all copies (Heidorn et al., 2011).

For applications beyond a laboratory setting, it is es-sential that marker genes (e.g. antibiotic resistance genes)do not remain in the genome. To achieve marker-freegenomic mutations, counterselection procedures havebeen developed for both Synechocystis sp. strain PCC 6803and Synechococcus sp. strain PCC 7002. For Synechocystissp. strain PCC 6803, the process requires two transfor-mation steps (Cheah et al., 2013). With the first transfor-mation, a cassette containing two marker genes, akanamycin resistance gene for positive selection and thetoxic mRNA interferase (mazF) gene for negative selection,is inserted into the genome via homologous recombina-tion. mazF is under the control of a nickel-inducible pro-moter. Successfully transformed cells are selected onnickel-free kanamycin-containing media and subjectedto a second round of transformation, in which the entirecassette inserted by the first transformation is replacedwith the gene of interest. Subsequent counterselection isperformed on kanamycin-free, nickel-containing medium.Cells that have not lost the marker gene cannot growbecause of the induction of mazF. A similar counter-selection method for Synechocystis sp. strain PCC 6803uses the Bacillus subtilis levan sucrase (sacB) gene as neg-ative selection marker (Lagarde et al., 2000). The disad-vantage of this counterselection system is the requirementfor a separate Glc-tolerant strain of Synechocystis sp. strainPCC 6803 as the chassis. An alternative strategy for Syn-echocystis sp. strain PCC 6803 is based on the Flippase/Flippase Recognition Target (FLP/FRT) recombinase





system from Saccharomyces cerevisiae rather than counter-selection. As with the above-mentioned counterselectionmethods, two transformation steps are needed (Tan et al.,2013). A successful counterselection procedure for Syn-echococcus sp. strain PCC 7002 is based on acrylatetoxicity and requires only one transformation step(Begemann et al., 2013). Deletion of the gene annotatedas acetyl-CoA ligase (acsA) through its replacementwith the DNA fragment of interest via homologousrecombination overcomes growth inhibition by acryl-ate. Thus, positive transformants are identified by theirability to grow on selective medium containing acryl-ate. To achieve expression of multiple heterologousgenes, the acsA gene can be reinserted into the ge-nome at a neutral site (e.g. a pseudogene annotatedas glycerol phosphate kinase; glpK). The organic acidcounterselection method is also potentially applicableto Synechocystis sp. strain PCC 6803, because AcsAactivity also confers acrylate sensitivity to this strain.

A major problem for the genetic manipulation of cya-nobacteria is their efficient system of restriction enzymesthat destroy foreign DNA introduced by any transfor-mation technique. One way to prevent DNA fragmen-tation is to ensure that the introduced DNA sequencecontains no sites that are recognized by the endogenousrestriction system. However, target sites differ betweencyanobacterial species, which is one reason why a shuttleor broad host vector that is maintained in one speciesmight be digested in another. A second approach is usedin conjugation, in which the helper plasmid can encodemethylases that protect against restriction enzymes com-monly present in many cyanobacteria (Vioque, 2007).

In conclusion, methods for genetic manipulation ofcyanobacteria have been established, but the number ofavailable tools is still limited. For example, a set of twointegrative vectors exist for Synechococcus sp. strain PCC7002 that recombine not with the chromosome but withendogenous plasmids (Xu et al., 2011). Because those canreach copy numbers of up to 50, high-level gene expres-sion is achieved. This elegant solution is not yet availablefor Synechocystis sp. strain PCC 6803. On the other hand,autonomously replicating plasmids are still missing forSynechococcus sp. strain PCC 7002, although a recentlydeveloped broad host range vector is a potential candi-date (Huang et al., 2010).

DESIGNING A SYNTHETIC BIODESALINATOR

Generation of a Salt-Free Biological Reservoir

The core of the proposed biodesalination process con-sists of the establishment of a salt-free (or low-salt) bio-logical reservoir within seawater that can serve as an ionexchanger. Most marine organisms already contain sucha reservoir because they actively exclude and remove saltfrom their bodies. Cyanobacteria employ a range of Na+

export proteins in their cell membrane (Fig. 3), all ofwhich are energized by the chemical energy carrier ATP.ATP powers Na+ export either directly through Na+-pumping ATPases, or indirectly through H+-pumping

ATPases, which generate a proton motive force thatdrives H+/Na+ antiport (Marin et al., 2004; Wiangnonet al., 2007). The ATP requirement offers an opportunityto halt Na+ export by depleting internal ATP storesusing the environmental manipulations detailed above(e.g. omitting photosynthetically efficient wavelengthsfrom the light spectrum, depleting phosphate, alteringpH, or chelating Mg2+, Fe2+, or other essential metals).Simply changing the growth system from an opensystem to a closed system once the culture has achievedhigh cell density may already rapidly deplete nutrientsupply and exhaust ATP reserves.

Designing Light-Powered Transport Modules

Once active Na+ export has come to a standstill, therewill be net Na+ influx into a cell until equilibrium with theexternal medium is reached. Further extraction of Na+

from the medium will then require an energy source. Toprevent renewal of Na+ export, the energy-harvestingsystem employed during this phase should not use ATPas an intermediate. Good candidates for ATP-independentlight-powered biological batteries are halorhodopsin (Hr)proteins. Hrs naturally occur in extremely salt-tolerant ar-chaea (haloarchaea) and are membrane-integral proteins ofthe rhodopsin superfamily that form a covalent bond withthe carotenoid-derived chromophore all-trans-retinal(Schobert and Lanyi, 1982; Klare et al., 2008). Absorp-tion of a photon with a defined optimal wavelengthinduces trans-cis isomerization of retinal, which triggersa catalytic photocycle of conformational changes in theprotein, resulting in the net import of one chloride perphoton into the cytoplasm. The turnover rates for light-activated ion pumps such as Hr are in the millisecondrange (Kolbe et al., 2000; Chizhov and Engelhard, 2001;Essen, 2002; Kouyama et al., 2010).

To date, several Hr proteins from different species havebeen characterized (Klare et al., 2008; Fu et al., 2012). TheHr from Natronomonas pharaonis (NpHr) has been clonedand successfully expressed in heterologous systems suchas E. coli, mammalian cells, and Xenopus laevis oocytes(Hohenfeld et al., 1999; Seki et al., 2007; Gradinaru et al.,2008). Expression of NpHr in X. laevis oocytes resulted ina light-dependent Cl2-inward current and consequently anegative shift in the membrane potential (Seki et al.,2007). The opportunity to artificially manipulate a cell’smembrane potential through NpHr in conjunction withlight-activated cation channels (e.g. channel rhodopsin)has been exploited in the field of optogenetics, achievingcontrol of action potentials in nerve cells with potentialmedicinal applications (Fenno et al., 2011; Zhang et al.,2011). We propose here that the negative membranepotential generated by Hr could also be used to drive theaccumulation of positively charged substances in cells.Thus, the expression of Hr could energize the uptake ofnutrients (e.g. Ca2+, Mg2+, K+, Fe2+), or toxic metals (e.g.Cd2+, Ni2+), into either plants or microorganisms, forbiofortification and bioremediation, respectively.

Expression of Hr in a high-density cyanobacterial cul-ture should remove both Cl2 and Na+ from surrounding


Amezaga et al.



seawater and thus provide a means for biodesalination.The observed Km values of Hrs for chloride uptake(approximately 25 mM for chloride; Duschl et al., 1990)are in an optimal range for this purpose. To increasethe speed of Na+ accumulation, the Na+ conductanceof the membrane might need to be enhanced by coex-pression of Na+-permeable channels or carriers with Hr(Fig. 3). Candidate proteins with different affinities andgating characteristics can be found in bacteria (Koishiet al., 2004), animals (Koopmann et al., 2006), and plants(Xue et al., 2011). The resulting light-powered salt ac-cumulator bypasses the endogenous energy metabolism(photosynthesis and respiration) and should thereforeremain functional even when increasing intracellular Na+

levels inhibit other metabolic functions of the host. A liv-ing cell would thus be transformed into a synthetic cell.

Ensuring Function and Robustness of theSynthetic Biodesalinator

Although technologies for environmental and geneticmanipulation of cyanobacteria are advancing fast andare predicted to enable realization of the core syntheticsalt accumulator, several additional challenges remainto be solved. First, only the protein part of Hr can be

heterologously expressed in cyanobacteria. The essentialall-trans-retinal is usually added as a supplement in thelaboratory, but this is not sustainable in a large-scaleprocess. Little is known about whether the enzymes thatproduce all-trans-retinal from b-carotene are present incyanobacteria. However, cyanobacteria as photosyn-thetic organisms already produce a wealth of carote-noids for light harvesting and photoprotection (Takaichiand Mochimaru, 2007); thus, engineering a syntheticpathway for the final enzymatic steps should not provetoo difficult. Second, progressive accumulation of NaClin the cells not only requires rapid osmotic adjustment ofthe cells (which most cyanobacteria are capable of), butalso threatens to lead to destabilization of membranesand proteins. It is therefore important that the cyano-bacterial strain is resistant to high salt concentrationsand that the heterologously expressed Hr and channelproteins are derived from naturally salt-tolerant species.Additional measures such as increasing the externalCa2+ concentration and altering lipid composition of themembrane should also be explored. Finally, even if thebiological materials are salt resistant, the biodesalinationprocess will need to be limited to a very narrow timewindow situated between the end of the growth phaseand the cell-removal phase. It is therefore essential toobtain control over the expression of introduced genes.

Figure 3. Na+ transport and its energization in different phases of the proposed desalination process. In the culture growthphase (left), the cells generate a low-salt reservoir inside the salty environment through active export of Na+ by endogenoustransport proteins (light gray circles) across the plasma membrane (PM). These are either directly fueled by ATP (Na+-ATPases)or, in the case of Na+/H+ antiporters, exploit the pH gradient established by H+-ATPases (dark gray circle). Na+ export from thecytoplasm (cyto) therefore relies on ATP and the proton motive force generated from light energy captured by photosystems(green box) and chemiosmosis (ATP-synthase, gray knob) in the thylakoid membrane (TM). In the desalination phase (right), Na+

export is halted through inhibition of photosynthetic ATP production. Instead, light energy is used directly by halorhodopsin(pink circle) to pump chloride into the cells. The resulting negative membrane potential (Vm) draws Na+ into the cell throughNa+-permeable channel proteins (gray box).





Gaining Control over Gene Expression in Cyanobacteria

Control over gene expression is exerted through pro-moter regions in the DNA, usually located immediatelyupstream of the gene, which are recognized by effectors(initiating transcription) as well as other regulatory pro-teins that link transcriptional activity to endogenous andenvironmental stimuli. Obtaining control over transgeneexpression in cyanobacteria requires the identificationand isolation of promoters that are responsive to thespecific triggers that will be used in the biotechnologicalprocess (environmental changes or supplements). Forexample, in the envisaged biodesalination process, pro-moters that are specifically active in the early stationaryphase of the culture could be cloned into the expressionvectors to activate the transgenes after the initial growthperiod. To ensure specificity and precise timing of genetranscription, the suitability of any candidate promoteras a biological switch needs to be tested in a range ofconditions and systems.

Promoter studies in cyanobacteria to date have pri-marily focused on characterizing native transcriptionalregulation in response to different environmental stimuli.Traditionally, Synechocystis sp. strain PCC 6803 wasstudied as a model for photosynthesis and circadianrhythm and several light-responsive promoters wereidentified, including the light-responsive (LR) promoter1(Marraccini et al., 1993) and the promoter of preproteintranslocase subunit (secA; Mazouni et al., 1998), as wellas the light-repressible promoter of PSI reaction centersubunits (psaAB; Muramatsu and Hihara, 2006), and thepromoter of light-repressed protein A homolog (lrtA;Imamura et al., 2004). More recently, cyanobacterialstudies have turned their focus to biotechnological ap-plications and numerous heavy metal-inducible pro-moters have been characterized (Peca et al., 2008; Blasiet al., 2012) as well as the copper-inducible promoterof plastocyanin (petE; Briggs et al., 1990; Ghassemianet al., 1994; Buikema and Haselkorn, 2001) and thecopper-repressible promoter of cytochrome c553 (petJ;Ghassemian et al., 1994). Furthermore, promoters tightlyregulated by nutrient availability have been character-ized, including the promoter of the sodium-dependentbicarbonate transporter (sbtA) regulated by inorganiccarbon availability (Wang et al., 2004) and the promoterof ferredoxin-nitrite reductase (nirA) regulated by nitro-gen source (Ivanikova et al., 2005; Qi et al., 2005).

The majority of studies characterizing cyanobacterialpromoter activity have been performed in the nativeorganisms. This poses a problem for transgenic appli-cations because of potential crosstalk and/or recombi-nation; therefore, in biotechnology, native promotersare generally avoided in favor of promoters fromclosely related organisms. The most common methodof gene regulation in bacteria is the lactose operonrepressor-operator (lacI-lacO); however, although thisworks well in some strains of cyanobacteria such asSynechococcus sp. strain PCC 7942 (Clerico et al., 2007), itis not suitable for others, including Synechocystis sp.strain PCC 6803 (Huang et al., 2010). Other promoters

that are well characterized in E. coli such as the so-calledPL and PR promoters of bacteriophage l have alsoshown poor functionality in cyanobacteria (Huanget al., 2010; Huang and Lindblad, 2013).

A range of different vectors and reporters have beenused to test promoter activity in cyanobacteria (Marracciniet al., 1993; Ivanikova et al., 2005; Peca et al., 2008; Huanget al., 2010; Xu et al., 2011; Blasi et al., 2012). In an attemptto standardize the characterization of promoter activityfor synthetic biology applications, a method was devel-oped in E. coli whereby promoter activity could be mea-sured relative to an in vivo reference promoter based onthe fluorescence intensities of GFP as a reporter (Kellyet al., 2009). The method was further developed using abroad host range vector derived from the so-called IncQplasmid, RSF1010, for promoter analysis in Synechocystissp. strain PCC 6803 (Huang et al., 2010). Because of thenature of the vector, this method can be applied to a widerange of organisms likely to include other cyanobacterialspecies.

In summary, some promoters regulated by differentstimuli have been identified and characterized in cya-nobacteria. For these to be suitable for biotechnologicalapplications, the activity of these promoters must becharacterized in nonnative settings, and standardizedmethods for characterization in cyanobacteria havebeen developed. At this stage, the availability of effec-tive biological switches is still a bottleneck for usage ofcyanobacteria as a chassis in synthetic biology and forbiotechnological applications.

STRATEGIES FOR CELL-WATER SEPARATION

Once biodesalination has occurred, efficient cell-waterseparation is the next step of the proposed process(Fig. 1). The notion of microorganisms as independentunicellular entities is continuously challenged by researchinto microbial biofilms (O’Toole et al., 2000). Neverthe-less, exploitation of photosynthetic microorganisms inwater treatment has focused predominantly on the useof unicellular microbial suspensions (i.e. planktonic cellsor suspended multispecies microflocs). Although theease of growth and maintenance favor use of planktoniccultures of cyanobacteria for biodesalination, the sepa-ration of such cells from the desalinated water duringdownstream processing without affecting the integ-rity of the cells and inadvertent release of sodiumchloride back into the desalinated water will be aneconomical and technical bottleneck in the bio-desalination process, as seen from previous attemptsat water treatment using photosynthetic microor-ganisms (Uduman et al., 2010; Lam and Lee, 2012;Olguín, 2012; Schlesinger et al., 2012). The difficultyin separating planktonic cyanobacterial cells fromaqueous suspensions stems from the fact that cellshave similar densities to water, cells behave likecolloidal particles because of the cell dimensions (fewmicrons), and cells possess charged surfaces thatstabilize cell suspensions.


Amezaga et al.



Metal Salts for Coagulation

The removal of photosynthetic microorganisms, es-pecially bloom-forming cyanobacterial strains such asMicrocystis aeruginosa and Nodularia sp., from water hasbeen studied in the context of water treatment processes.Therefore, the cell-liquid separation techniques have bor-rowed heavily from wastewater treatment procedures,although centrifugation and filtration are employed whenproduct quality, especially of high-value chemicals, is tobe ensured. Nevertheless, coagulation- and flocculation-based processes are considered to be more energy efficientand cost-effective than centrifugation and filtration(Uduman et al., 2010; Lam and Lee, 2012). Inorganic metalsalts such as aluminum sulfate (4.8 to 5.8 mg/L and 65 to70 mg/L; Chow et al., 1999; Drikas et al., 2001, respec-tively), ferric chloride (30 mg/L; Chow et al., 1998), andpolyaluminum chloride (4 mg/L; Sun et al., 2013) areeffective at separating out up to 99% of cyanobacteriafrom water. Aggregation of cells with addition of metalsalts is mediated by the neutralization of surface charges(Lam and Lee, 2012). In these studies, the added coagu-lants did not affect the cell membrane integrity or causetoxin release from the cells during flocculation. However,extensive cell damage and release of intracellular com-ponents can occur during floc storage and recycling,downstream of the flocculation process (Sun et al., 2013).A related issue is that coagulation is normally operated atan acidic pH during water treatment, which photosyn-thetic organisms may not tolerate (Kim et al., 2011a).

Polyionic Polymers for Coagulation

Formation of aggregates through the use of syntheticand organic polymers (i.e. polymer bridging) has beeninvestigated as an alternative to the use of metal salts,with some success. Synthetic cationic polymers such aspolyethylenimine (20 to 30 mg/L; Zeleznik et al., 2002;Arrington et al., 2003), polyacrylamide (3 mg/L; Jan�culaet al., 2011) and the polyacrylamide-based Praestol(1 mg/L; Pushparaj et al., 1993) are able to flocculatecyanobacterial cells with between 80% and 90% efficiencyof cell removal. Praestol did not affect cell membraneintegrity, but polyethylenimine was shown to increasecell permeability. The effect of polyacrylamide on the cellviability was not tested. In addition to synthetic poly-mers, organic flocculants such as clay and chitosan en-hance the flocculation ability of cyanobacteria (Divakaranand Sivasankara Pillai, 2002; Pan et al., 2006a, 2006b;Verspagen et al., 2006; Zou et al., 2006; Liu et al., 2010).Although no adverse effect on cell membrane integrityhas been demonstrated with the addition of chitosan, theuse of clay and chemically modified clay, especiallychitosan-modified kaolinite, results in widespread deathand lysis of cyanobacterial cells (Shao et al., 2012). Be-cause the conditions during floc formation such as tem-perature, ionic strength of the suspension medium, pH,strain type, and cell concentration differ between studies,the efficiency of the polymers in cell removal cannot bedirectly compared. Moreover, the efficiency of cell-liquid

separation using flocculation-based technologies is notconsistent. It depends to a great extent on the surfacecharacteristics of the suspended cells and the polymerspresent in the environment. These can be either naturalorganic matter or polymers produced by the cells duringgrowth (i.e.EPSs, also known as algogenic organic matter;Henderson et al., 2010; Teixeira et al., 2010).

Microbial EPSs for Coagulation

Microbial EPSs are categorized in two separate frac-tions based on proximity to the cell surface. EPSs posi-tioned near the cell surface by noncovalent interactionsare termed bound EPSs, and those that are secreted intothe culture medium are called free or released EPSs(Eboigbodin and Biggs, 2008). The aggregation of cellswithin a biofilm is known to be aided by the favorableinteractions between physicochemistry of the cell surfaceand the EPS (Karunakaran and Biggs, 2011). However,when using coagulants, especially polyvalent metal salts,to induce aggregation, EPSs of Aphanothece halophyticaand Microcystis aeruginosa increase coagulant demand(Takaara et al., 2007, 2010; Chen et al., 2009, 2010;Henderson et al., 2010). On the other hand, the presenceof EPSs, without the use of metal salts, can induce ag-gregation. The bioflocculation of kaolinite using releasedEPSs isolated from cultures of Phormidium sp., Anabaenacircularis, Lynbyga sp., andMicrocoleus sp. was previouslyreported (Levy et al. 1990, 1992; Chen et al., 2011). Therole of EPSs in flocculation was recently proposed forSynechocystis sp. strain PCC 6803 (Jittawuttipoka et al.,2013). Moreover, bioflocculant activity is not limited toreleased cyanobacterial EPSs (Taniguchi et al., 2005; Kimet al., 2011a; Nie et al., 2011). Interestingly, the boundEPSs of cyanobacterial and heterotrophic cells have alsobeen indicated to aid flocculation. In species such asArthrospira plantensis, T. tenuis, and Desulfovibrio oxy-clinae, autoflocculation is induced when the cells areexposed to environmental stress (Sigalevich et al., 2000;Silva and Silva, 2007; Markou et al., 2012). Acinetobactercalcoaceticus, a water isolate, will not only autoflocculatebut will also enhance the flocculation ability of otherbacteria (Simões et al., 2008). In addition, the bio-flocculation of algae using EPSs does not affect cellmembrane integrity (Lee et al., 2009; Kim et al., 2011a).

In conclusion, the harvesting of biomass withoutaffecting the integrity of the cells is an importantarea of research within industrial biotechnology. Bio-flocculation of cells is a balance between the physico-chemical properties of the cell surface and EPSs, andcould be a preferable alternative to chemical coagu-lants. However, to facilitate biomass harvesting usingbioflocculants at the industrial scale, a rigorous studyof the cell surface characteristics and EPS productionof the cells under relevant operating conditions has tobe carried out, especially because the cell surface andEPSs have been shown to be affected by the environ-mental conditions (Eboigbodin et al., 2006, 2007;Mukherjee et al., 2012). Overall, there is an urgent needfor in-depth characterization of surface properties and of





EPSs in photosynthetic organisms so that suitable cell-water separation technologies can be developed.

DESIGN OF AN INTEGRATED PROCESS

The overall aims of municipal wastewater treatmentplants and water treatment plants are to protect publichealth in a manner compatible with environmental, eco-nomic, social, and political concerns. Wastewater treat-ment commonly utilizes biological processes relying onmicroorganisms to take up dissolved organic matter andnutrients. These processes take advantage of the fact thatmicroorganisms are relatively easy to remove throughsettling or filtration. Biological treatment technologiesdeployed in wastewater treatment include the activatedsludge process (aerobic suspended growth), trickling fil-ters (as well as other attached-growth biological filters),and membrane bioreactors (membrane filtration com-bined with a suspended growth bioreactor). More ad-vanced configurations of the activated sludge process,incorporating aerobic and anoxic zones, can be operatedfor nutrient removal. There are increasing regulatorypressures, such as those in the EU urban wastewatertreatment directive (European Union, 1991), to limit ni-trate and phosphate contents with the aim to protectdownstream aquatic ecosystems from eutrophication.This is achieved through nitrification, denitrification, andphosphate uptake by different communities of bacteria.Other biological technologies used in wastewater treat-ment plants are aerobic lagoons and various suspended-and fixed-growth anaerobic processes (including a rangeof anaerobic digester and anaerobic filter designs).

Reactors designed to promote viability, functionality,and high concentrations of photosynthetic organismsmay differ significantly from those used in biologicalwastewater treatment, even if both are based on princi-ples of attached and suspended growth. Evidently, lightis a key parameter and reactors used to grow algae mayprove more suitable in this respect. Many photo-bioreactor designs are only used at the laboratory scaleand recent advances in light-emitting diode technologiesoffer an opportunity to efficiently supply the requisitewavelengths of light for photosynthesis. However, at fullscale, this becomes less feasible in terms of operationaland capital costs, with a key challenge of providing andregulating light exposure to photosynthetic organisms.Large-scale open lagoons are an appropriate system toachieve this. In common with many engineered algalcultures, these are more favorable in locations with year-round high solar radiation and temperature (Su et al.,2011). Nonetheless, many design improvements are stillneeded in order to improve robustness, reduce energyconsumption, and optimize growth conditions for large-scale production of photoautotrophs. Providing a feedwith the appropriate nutrient profile and suitable tem-perature, and mitigating against interference from otherindigenous microorganisms are other key challengeslinked to a transition from growing photosynthetic or-ganisms at the laboratory scale to the industrial scale. Ofthe nutrients required for photoautotrophic growth,

carbon dioxide is considered as the most significant,because of the high proportion (approximately 50% ofdry weight) of carbon in the biomass of photoautotro-phic organisms (Kim et al., 2011b). Large-scale growth ofphotoautotrophic organisms relies upon huge amountsof carbon dioxide, which must be delivered in an energy-efficient manner. At the laboratory scale, this can beeasily provided by sparging with air and/or carbon di-oxide. Bubbleless gas-transfer membranes, widely usedin the food industry, show promise for larger-scale de-livery of carbon dioxide (Kim et al., 2011b). Overall, inorder to achieve improved reactor design, it is critical tobetter understand the kinetics of nutrient acquisition andphoton capture by relevant organisms, so that theirgrowth and rates of photosynthesis can be properlycontrolled.

A crucial operational issue common to both waste-water treatment and growth of photosynthetic orga-nisms is delivering a sustainable and cost-effectivedisposal or reuse route for the large volumes of biomassthat will inevitably be produced. Promising avenues toachieve this exist. Notably, these include anaerobic di-gestion, biofuel production, or utilization as a feedsubstrate in aquaculture. With respect to biofuel pro-duction, genetically modified (GM) strains of Synecho-cystis sp. strain PCC 6803 have been grown that secreteenergy-rich fatty acids (Liu et al., 2011). Experiencefrom disposal of sewage sludge shows there are anumber of challenges that will need to be overcomebefore reuse of waste biomass from photosynthetic or-ganisms will become viable. These include effectivelow-energy dewatering and complying with the rele-vant legislation for reuse and disposal of biosolids,which is likely to be a particular issue for GM biomass.The impact of residual salt on downstream reuse ap-plications also requires consideration. Although anaer-obically digesting biomass has the major benefit ofgenerating methane (a potential energy source), algalsludge tends to be of relatively low biodegradabilityand methane yield (Bond et al., 2012). In such situations,pretreatment or hydrolysis, to increase biodegrad-ability, and/or codigestion with a complementaryfeed source are possible methods to improve digesterperformance.

The design of clarifiers for effective separation ofphotosynthetic organisms from water is another im-portant issue to consider when moving from a labora-tory scale to a full scale of operation. Sedimentation andflotation are two economically viable cell-liquid sepa-ration techniques typically employed in water treat-ment plants. Both approaches require coagulation andefficient floc formation to achieve high separation effi-ciencies. Sedimentation has the advantage of low capitalexpenditure and low energy consumption during op-eration compared with flotation. However, the ability ofcyanobacteria such as Synechocystis sp. strain PCC 6803and Synechococcus sp. strain PCC 7002 to rise to thesurface of an open container (Fig. 4) suggests that flo-tation strategies such as dissolved air flotation can helpachieve high separation efficiencies rapidly.


Amezaga et al.



ASSESSMENT OF RISK AND PUBLIC ACCEPTANCE

Any application of biodesalination technology hasnumerous health and environmental protection issuesthat must be addressed during the design, construction,and operation of the facility (World Health Organization,2007). In addition, the use of synthetic biological ap-plications, particularly involving cyanobacteria withits known toxicity risks (Hunter et al., 2012), brings with itthe risk of low social acceptance (Bubela et al., 2012).Indeed, the general public has historically been skepticalabout adopting alternative water sources in general(Dolnicar et al., 2010) and proposed schemes have evenbeen abandoned because of a lack of public acceptance(Po et al., 2003; Hurlimann and McKay, 2007; Hurlimannand Dolnicar, 2010). Much research has been undertakeninto public acceptance of recycled water, particularly incountries such as Australia, where serious droughts, withtheir accompanying severe water restrictions, have led tothe search for alternative water supplies. More recently,researchers have begun to also investigate public accep-tance of desalinated water and have discovered differentdegrees of acceptance, for both recycled and desalinatedwater, depending upon the particular use intended(Hurlimann and Dolnicar, 2011). Greater acceptance ofdesalinated water, as opposed to recycled water, has beenfound for close-to-body uses, whereas recycled water ispreferred for uses not close to the body (e.g. irrigation orindustrial cooling; Dolnicar and Schäfer, 2009; Dolnicaret al., 2011).Factors such as education, age, knowledge, income,

and sex influence acceptance levels of recycled water(Dolnicar and Schäfer, 2009). In general, the more formalthe education received by a person, the greater theirknowledge about recycled water and the higher theprobability that they will accept it (Sims and Baumann,1974). Related to this factor, Baumann (1983) found thatthe better educated respondents had a greater faith inscience and technology and therefore a higher acceptance.Similarly, Marks (2006) argues that effective public con-sultation promotes greater trust in those responsible forthe assessment and management of risks, and Po et al.(2003) ascribe the success of a number of water reuseprojects to a great emphasis on public involvement andeducation. As far as desalination is concerned, it has beennoted that the knowledge level concerning the tech-nology is relatively low (Dolnicar et al., 2011); thus,increasing the public’s knowledge could increase ac-ceptance levels. Dolnicar et al. (2010) looked specificallyat how the provision of information about alternativewater supplies affected public perception. They con-cluded that hesitance to embrace such water is primarilydriven by water quality concerns, but providing peoplewith basic information about recycled and desalinatedwater increased their likelihood of using these alterna-tive supplies.In addition to the general skepticism over the use of

desalinated water, the use of synthetic biological ap-plications in the field of biodesalination, particularlythose involving GM cyanobacteria with their inherent

risks (Henley et al., 2013), increases the danger of lowsocial acceptance. Historically, public opinion on whatmay be viewed as the (re)design of nature and themerging of biology with engineering has been negative(Bubela et al., 2012). As with the introduction ofrecycled and desalinated water, however, the provi-sion of accurate information on the benefits and risksof the technology in the early stages of any proposed

Figure 4. Cell-water separation can take advantage of the ability ofcyanobacteria to float. Visual appearance of initially mixed cultures ofcyanobacteria strains Synechocystis sp. strain PCC 6803 (top) andSynechococcus sp. strain PCC 7002 (bottom) left under ambient lab-oratory light (8 6 2 mM) for 24 h (n = 3). [See online article for colorversion of this figure.]





project is believed to be critical, particularly concern-ing the image portrayed by the news media, which canhave an adverse influence on acceptance levels (Bubelaet al., 2012). Notwithstanding this, Christoph et al.(2008) concluded that educating consumers does notnecessarily result in greater support for genetic modi-fication because increased knowledge does not auto-matically imply support.

Despite the importance of public opinion to the suc-cess of emerging technologies, there remains a paucityof studies in the literature on public perceptions ofsynthetic biology. The majority of research has beenundertaken on social acceptance of GM food products(Costa-Font et al., 2008; Siegrist, 2008; Dannenberg,2009). It is frequently argued that consumer rejection ofsuch foods is the result of their introduction withoutany perceived benefits to consumers, together with theportrayed risks of genetically modified organisms(GMOs) to the environment (Frewer et al., 2004). Otherfactors such as ethical and moral considerations andtrust in both the scientists conducting the research andthe regulatory system are also important determinantsof consumer acceptance or rejection of the technology(Frewer, 2003; Siegrist, 2008). In a study by Magnussonand Koivisto Hursti (2002), it was discovered that ageand sex, together with level of education, had an impacton likely acceptance of GM foods, with males andyounger respondents generally being more positive.Meanwhile, Prokop et al. (2013) discovered that diseaserisk resulted in significantly more negative attitudes to-ward GM products. However, with current stringentregulations governing the use of synthetic biologicalapplications, such concerns should be minimized, espe-cially if the public is kept reliably informed from theearly stages of development.

One of the key considerations in the application of thebiodesalination technology concerns potential locations.Issues of saline waters, and the requirement for desali-nation to augment supplies, are well known in the GulfStates and South America, where conventional desali-nation plants already exist (Dawoud, 2005). Social ac-ceptance of emerging technologies has been shown tovary between countries. In particular, the experience ofserious drought and water restrictions in Australia hasled to less resistance to recycled or desalinated water inrecent years (Dolnicar and Schäfer, 2009,) suggestingthat public opinions are affected by personal experi-ences. Historically, developing countries were less op-posed to the concept of genetic modification. However,Frewer (2003) noted an increasing resistance to the in-troduction of GM foods in developing countries as aresult of activity of national government organizationsthat oppose the implementation of genetic technologies inagriculture. Meanwhile, studies in Germany (Christophet al., 2008) and Sweden (Magnusson and KoivistroHursti, 2002) found strong negative tendencies to theacceptance of genetic modification, with the main con-cern being uncertainty about possible long-term effectsto the environment and human health. Acceptability wasgreater toward applications involving nonfood products,

however, because they are seen to be more beneficial,less risky, and ethically correct, a point also noted bySorgo et al. (2012).

A biodesalination process based on GM cyanobacte-ria will present multiple challenges from the point ofview of social and regulatory acceptance. It has clearlymore chance of success in countries in which desalina-tion is already an accepted practice, and where GMOsare not seen as a threat by both government and pop-ulation. The process will have to ensure that it fulfills allsafety requirements for GMO approval. It will also haveto prove that there is no danger coming from the use ofcyanobacteria and to actively deal with potential nega-tive perceptions as a result of toxin generation. Conse-quently, it seems advisable to explore initially combineduses of the low salinity water and biomass in productivesystems designed for saline arid environments.

CONCLUSION AND OUTLOOK

This article examined, using the specific example ofbiodesalination, the challenges and opportunities as-sociated with applications of cyanobacteria in watertreatment, many of which are pertinent to other bio-technologies. The key part of the conceptualized bio-desalination process is to employ a low-salt biologicalreservoir within the cyanobacteria as an ion exchanger.Uptake of salt into these reservoirs would then be me-diated by genetic and/or environmental manipulationof the cyanobacteria. As exemplified by Synechocystis sp.strain PCC 6803 and Synechococcus sp. strain PCC7002, cyanobacteria have a number of attributes thatmake them attractive for such applications, becausethey are fast-growing, tolerant of a range of salt con-centrations, and amenable to genetic transformation.Furthermore, because the primary metabolism of cyano-bacteria is based upon photosynthesis, nutrient require-ments are minimal and active salt export during growth ispowered by sunlight. Solar radiation can also be used toenergize subsequent salt accumulation through expres-sion of retinal ion pumps such as Hr. Protocols for geneticmanipulation of cyanobacteria through natural transfor-mation and conjugation have been developed. As is thecase in other biotechnological processes, biodesalinationrequires efficient separation of cells from water. Coagu-lation is a suitable method, because this can remove up to99% of cyanobacteria and chitosan flocculants have noadverse impact on viability of cyanobacteria. The designand operation of an integrated biodesalination process islikely to build on knowledge of both algal bioreactors andwastewater treatment processes.

Notwithstanding these opportunities, challenges needto be overcome at each stage of the proposed bio-desalination process. Further research is needed to elu-cidate the impact of environmental factors, includingpH, temperature, and nutrients, on salt transport in cy-anobacteria. A major bottleneck for easy genetic ma-nipulation is the limited availability of vector backbonesthat enable flexible rearrangement of essential elements,and of robust promoters that can operate as biological


Amezaga et al.



switches in nonnative settings. Furthermore, separationof planktonic cyanobacteria fromwater is difficult becauseof their low density and molecular size, and the presenceof EPSs can have contradictory effects on aggregation.Consequently, to fully optimize separation, more work isneeded to characterize the surface properties of both cy-anobacteria and EPSs. Finally, the use of synthetic bio-logical applications to produce recycled water brings therisk of low social acceptance, although this varies geo-graphically and may increase with further education.

ACKNOWLEDGMENTS

The authors of this article joined forces to develop methods for biodesali-nation after a Sandpit event (Water For All Challenge, 2010) organized by theEngineering and Physical Sciences Research Council.

Received December 10, 2013; accepted March 5, 2014; published March 7,2014.

LITERATURE CITED

Allakhverdiev SI, Murata N (2008) Salt stress inhibits photosystems II andI in cyanobacteria. Photosynth Res 98: 529–539

Altermann W, Kazmierczak J (2003) Archean microfossils: a reappraisal ofearly life on Earth. Res Microbiol 154: 611–617

Apte SK, Reddy BR, Thomas J (1987) Relationship between sodium influxand salt tolerance of nitrogen-fixing cyanobacteria. Appl Environ Mi-crobiol 53: 1934–1939

Apte SK, Thomas J (1983) Sodium transport in filamentous nitrogen fixingcyanobacteria. J Biosci 5: 225–233

Apte SK, Thomas J (1997) Possible amelioration of coastal soil salinityusing halotolerant nitrogen-fixing cyanobacteria. Plant Soil 189: 205–211

Arrington SA, Zeleznik MJ, Ott DW, Ju LK (2003) Effects of poly-ethyleneimine on cyanobacterium Anabaena flos-aquae during cell floc-culation and flotation. Enzyme Microb Technol 32: 290–293

Baebprasert W, Karnchanatat A, Lindbland P, Incharoensakdi A (2011)Na+-stimulated nitrate uptake with increased activity under osmoticupshift in Synechocystis sp. strain PCC 6803. World J Microbiol Bio-technol 27: 2467–2473

Barten R, Lill H (1995) DNA-uptake in the naturally competent cyano-bacterium, Synechocystis sp. PCC-6803. FEMS Microbiol Lett 129: 83–88

Baumann DD (1983) Social acceptance of water reuse. Appl Geogr 3: 79–84Begemann MB, Zess EK, Walters EM, Schmitt EF, Markley AL, Pfleger BF

(2013) An organic acid based counter selection system for cyanobacteria.PLoS ONE 8: e76594

Bennett A, Bogorad L (1973) Complementary chromatic adaptation in afilamentous blue-green alga. J Cell Biol 58: 419–435

Bhargava S, Saxena RK, Pandey PK, Bisen PS (2003) Mutational engi-neering of the cyanobacterium Nostoc muscorum for resistance to growth-inhibitory action of LiCl and NaCl. Curr Microbiol 47: 5–11

Blasi B, Peca L, Vass I, Kós PB (2012) Characterization of stress responsesof heavy metal and metalloid inducible promoters in SynechocystisPCC6803. J Microbiol Biotechnol 22: 166–169

Blumwald E, Mehlhorn RJ, Packer L (1983) Ionic osmoregulation during salt ad-aptation of the cyanobacterium Synechococcus 6311. Plant Physiol 73: 377–380

Bond T, Brouckaert CJ, Foxon KM, Buckley CA (2012) A critical review ofexperimental and predicted methane generation from anaerobic codi-gestion. Water Sci Technol 65: 183–189

Briggs LM, Pecoraro VL, McIntosh L (1990) Copper-induced expression,cloning, and regulatory studies of the plastocyanin gene from the cya-nobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 15: 633–642

Bruns BU, Briggs WR, Grossman AR (1989) Molecular characterization of phyco-bilisome regulatory mutants of Fremyella-Diplosiphon. J Bacteriol 171: 901–908

Bubela T, Hagen G, Einsiedel E (2012) Synthetic biology confronts publicsand policy makers: challenges for communication, regulation andcommercialization. Trends Biotechnol 30: 132–137

Buikema WJ, Haselkorn R (2001) Expression of the Anabaena hetR genefrom a copper-regulated promoter leads to heterocyst differentiationunder repressing conditions. Proc Natl Acad Sci USA 98: 2729–2734

Cheah YE, Albers SC, Peebles CAM (2013) A novel counter-selectionmethod for markerless genetic modification in Synechocystis sp. PCC6803. Biotechnol Prog 29: 23–30

Chen L, Li P, Liu Z, Jiao Q (2009) The released polysaccharide of the cy-anobacterium Aphanothece halophytica inhibits flocculation of the algawith ferric chloride. J Appl Phycol 21: 327–331

Chen L, Li T, Guan L, Zhou Y, Li P (2011) Flocculating activities of poly-saccharides released from the marine mat-forming cyanobacteria Mi-crocoleus and Lyngbya. Aquat Biol 11: 243–248

Chen L, Men X, Ma M, Li P, Jiao Q, Lu S, Kong F, Wu S (2010) Polysac-charide release by Aphanothece halophytica inhibits cyanobacteria/clayflocculation. J Phycol 46: 417–423

Chizhov I, Engelhard M (2001) Temperature and halide dependence of thephotocycle of halorhodopsin from Natronobacterium pharaonis. Biophys J81: 1600–1612

Chow CWK, Drikas M, House J, Burch MD, Velzeboer RMA (1999) Theimpact of conventional water treatment processes on cells of the cya-nobacterium Microcystis aeruginosa. Water Res 33: 3253–3262

Chow CWK, House J, Velzeboer RMA, Drikas M, Burch MD, SteffensenDA (1998) The effect of ferric chloride flocculation on cyanobacterialcells. Water Res 32: 808–814

Christoph IB, Bruhn M, Roosen J (2008) Knowledge, attitudes towardsand acceptability of genetic modification in Germany. Appetite 51:58–68

Clerico EM, Ditty JL, Golden SS (2007) Specialized techniques for site-directed mutagenesis in cyanobacteria. Methods Mol Biol 362: 155–171

Costa-Font M, Gil JM, Traill WB (2008) Consumer acceptance, valuation ofand attitudes towards genetically modified food: review and implica-tions for food policy. Food Policy 33: 99–111

Dannenberg A (2009) The dispersion and development of consumer pref-erences for genetically modified food – a meta-analysis. Ecol Econ 68:2182–2192

Dawoud MA (2005) The role of desalination in augmentation of watersupply in GCC countries. Desalination 186: 187–198

Divakaran R, Sivasankara Pillai VN (2002) Flocculation of algae usingchitosan. J Appl Phycol 14: 419–422

Dolnicar S, Schäfer AI (2009) Desalinated versus recycled water: publicperceptions and profiles of the accepters. J Environ Manage 90: 888–900

Dolnicar S, Hurlimann A, Grün B (2011) What affects public acceptance ofrecycled and desalinated water? Water Res 45: 933–943

Dolnicar S, Hurlimann A, Nghiem LD (2010) The effect of information onpublic acceptance—the case of water from alternative sources. J EnvironManage 91: 1288–1293

Drikas M, Chow CWK, House J, Burch MD (2001) Using coagulation,flocculation, and settling to remote toxic cyanobacteria. J Am WaterWorks Assoc 93: 100–111

Duschl A, Lanyi JK, Zimányi L (1990) Properties and photochemistry of ahalorhodopsin from the haloalkalophile, Natronobacterium pharaonis. JBiol Chem 265: 1261–1267

Eboigbodin KE, Biggs CA (2008) Characterization of the extracellularpolymeric substances produced by Escherichia coli using infrared spec-troscopic, proteomic, and aggregation studies. Biomacromolecules 9:686–695

Eboigbodin KE, Newton JR, Routh AF, Biggs CA (2006) Bacterial quorumsensing and cell surface electrokinetic properties. Appl Microbiol Bio-technol 73: 669–675

Eboigbodin KE, Ojeda JJ, Biggs CA (2007) Investigating the surfaceproperties of Escherichia coli under glucose controlled conditions and itseffect on aggregation. Langmuir 23: 6691–6697

Engelbrecht F, Marin K, Hagemann M (1999) Expression of the ggpS gene,involved in osmolyte synthesis in the marine cyanobacterium Synecho-coccus sp. strain PCC 7002, revealed regulatory differences between thisstrain and the freshwater strain Synechocystis sp. strain PCC 6803. ApplEnviron Microbiol 65: 4822–4829

Espie GS, Miller AG, Canvin DT (1988) Characterization of the Na-requirement in cyanobacterial photosynthesis. Plant Physiol 88: 757–763

Essen LO (2002) Halorhodopsin: light-driven ion pumping made simple?Curr Opin Struct Biol 12: 516–522

Essich E, Stevens SE Jr, Porter RD (1990) Chromosomal transformation in thecyanobacterium Agmenellum quadruplicatum. J Bacteriol 172: 1916–1922

European Union (1991) Council Directive 91/271/EEC of May 21, 1991concerning urban waste-water treatment. EUR-Lex, Access to EuropeanLaw. http://eur-lex.europa.eu/ (March 17, 2014)




http://eur-lex.europa.eu/


Fenno L, Yizhar O, Deisseroth K (2011) The development and applicationof optogenetics. Annu Rev Neurosci 34: 389–412

Frewer L (2003) Societal issues and public attitudes towards geneticallymodified foods. Trends Food Sci Technol 14: 319–332

Frewer L, Lassen J, Kettlitz B, Scholderer J, Beekman V, Berdal KG (2004)Societal aspects of genetically modified foods. Food Chem Toxicol 42:1181–1193

Frigaard NU, Sakuragi Y, Bryant DA (2004) Gene inactivation in the cya-nobacterium Synechococcus sp. PCC 7002 and the green sulfur bacte-rium Chlorobium tepidum using in vitro-made DNA constructs andnatural transformation. Methods Mol Biol 274: 325–340

Fu HY, Chang YN, Jheng MJ, Yang CS (2012) Ser(262) determines thechloride-dependent colour tuning of a new halorhodopsin from Hal-oquadratum walsbyi. Biosci Rep 32: 501–509

Fulda S, Huckauf J, Schoor A, Hagemann M (1999) Analysis of stress re-sponses in the cyanobacterial strains Synechococcus sp. PCC 7942, Syn-echocystis sp. PCC 6803, and Synechococcus sp. PCC 7814: osmolyteaccumulation and stress protein synthesis. J Plant Physiol 154: 240–249

Ghassemian M, Wong B, Ferreira F, Markley JL, Straus NA (1994) Clon-ing, sequencing and transcriptional studies of the genes for cytochromec-553 and plastocyanin from Anabaena sp. PCC 7120. Microbiology 140:1151–1159

Gradinaru V, Thompson KR, Deisseroth K (2008) eNpHR: a Natronomonashalorhodopsin enhanced for optogenetic applications. Brain Cell Biol 36:129–139

Grigorieva G, Shestakov S (1982) Transformation in the cyanobacteriumSynechocystis sp. 6803. FEMS Microbiol Lett 13: 367–370

Hagemann M (2010) Molecular biology of cyanobacterial salt acclimation.FEMS Microbiol Rev 35: 87–123

Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjö K,Lindblad P (2011) Synthetic biology in cyanobacteria engineering andanalyzing novel functions. Methods Enzymol 497: 539–579

Henderson RK, Parsons SA, Jefferson B (2010) The impact of differing celland algogenic organic matter (AOM) characteristics on the coagulationand flotation of algae. Water Res 44: 3617–3624

Henley WJ, Litaker RW, Novoveská L, Duke CS, Quemada HD, Sayre RT(2013) Initial risk assessment of genetically modified (GM) microalgaefor commodity-scale biofuel cultivation. Algal Res 2: 66–77

Hohenfeld IP, Wegener AA, Engelhard M (1999) Purification of histidinetagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sen-sory rhodopsin II functionally expressed in Escherichia coli. FEBS Lett442: 198–202

Huang HH, Camsund D, Lindblad P, Heidorn T (2010) Design and char-acterization of molecular tools for a synthetic biology approach towardsdeveloping cyanobacterial biotechnology. Nucleic Acids Res 38: 2577–2593

Huang HH, Lindblad P (2013) Wide-dynamic-range promoters engineeredfor cyanobacteria. J Biol Eng 7: 10–21

Huflejt ME, Tremolieres A, Pineau B, Lang JK, Hatheway J, Packer L(1990) Changes in membrane lipid composition during saline growth ofthe fresh water cyanobacterium Synechococcus 6311. Plant Physiol 94:1512–1521

Hunter PD, Hanley N, Czajkowski M, Mearns K, Tyler AN, Carvalho L,Codd GA (2012) The effect of risk perception on public preferences andwillingness to pay for reductions in the health risks posed by toxic cy-anobacterial blooms. Sci Total Environ 426: 32–44

Hurlimann A, Dolnicar S (2010) When public opposition defeats alterna-tive water projects - the case of Toowoomba Australia. Water Res 44:287–297

Hurlimann A, Dolnicar S (2011) Voluntary relocation – an exploration ofAustralian attitudes in the context of drought, recycled and desalinatedwater. Glob Environ Change 21: 1084–1094

Hurlimann A, McKay J (2007) Urban Australians using recycled water fordomestic non-potable use—an evaluation of the attributes price, salti-ness, colour and odour using conjoint analysis. J Environ Manage 83: 93–104

Imamura S, Asayama M, Shirai M (2004) In vitro transcription analysisby reconstituted cyanobacterial RNA polymerase: roles of group1 and 2 sigma factors and a core subunit, RpoC2. Genes Cells 9: 1175–1187

Inthorn D, Nagase H, Isaji Y, Hirata K, Miyamoto K (1996) Removal ofcadmium from aqueous solutions by the filamentous cyanobacteriumTolypothrix tenuis. J Ferment Bioeng 82: 580–584

Ivanikova NV, McKay RML, Bullerjahn GS (2005) Construction andcharacterization of a cyanobacterial bioreporter capable of assessingnitrate assimilatory capacity in freshwaters. Limnol Oceanogr Methods3: 86–93

Jan�cula D, Maršálková E, Maršálek B (2011) Organic flocculants for theremoval of phytoplankton biomass. Aquacult Int 19: 1207–1216

Jittawuttipoka T, Planchon M, Spalla O, Benzerara K, Guyot F, Cassier-Chauvat C, Chauvat F (2013) Multidisciplinary evidences that Syn-echocystis PCC6803 exopolysaccharides operate in cell sedimentationand protection against salt and metal stresses. PLoS ONE 8: e55564

Karunakaran E, Biggs CA (2011) Mechanisms of Bacillus cereus biofilmformation: an investigation of the physicochemical characteristics of cellsurfaces and extracellular proteins. Appl Microbiol Biotechnol 89: 1161–1175

Kazusa DNA Research Institute (2013) CyanoBase. http://genome.microbedb.jp/cyanobase (accessed December 10, 2013)

Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, deMora K, Glieberman AL, Monie DD, Endy D (2009) Measuring theactivity of BioBrick promoters using an in vivo reference standard. J BiolEng 3: 4

Kim DG, La HJ, Ahn CY, Park YH, Oh HM (2011a) Harvest of Scenedesmussp. with bioflocculant and reuse of culture medium for subsequent high-density cultures. Bioresour Technol 102: 3163–3168

Kim HW, Marcus AK, Shin JH, Rittmann BE (2011b) Advanced control forphotoautotrophic growth and CO2-utilization efficiency using a mem-brane carbonation photobioreactor (MCPBR). Environ Sci Technol 45:5032–5038

Klare JP, Chizhov I, Engelhard M (2008) Microbial rhodopsins: scaffoldsfor ion pumps, channels, and sensors. Results Probl Cell Differ 45: 73–122

Koishi R, Xu HX, Ren DJ, Navarro B, Spiller BW, Shi Q, Clapham DE(2004) A superfamily of voltage-gated sodium channels in bacteria. J BiolChem 279: 9532–9538

Koksharova O, Wolk C (2002) Genetic tools for cyanobacteria. 58: 123–137Kolbe M, Besir H, Essen LO, Oesterhelt D (2000) Structure of the light-

driven chloride pump halorhodopsin at 1.8 A resolution. Science 288:1390–1396

Koopmann TT, Bezzina CR, Wilde AA (2006) Voltage-gated sodiumchannels: action players with many faces. Ann Med 38: 472–482

Kouyama T, Kanada S, Takeguchi Y, Narusawa A, Murakami M, Ihara K(2010) Crystal structure of the light-driven chloride pump halorhodop-sin from Natronomonas pharaonis. J Mol Biol 396: 564–579

Kufryk GI, Sachet M, Schmetterer G, Vermaas WFJ (2002) Transfor-mation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool forgenetic mapping: optimization of efficiency. FEMS Microbiol Lett 206:215–219

Labarre J, Chauvat F, Thuriaux P (1989) Insertional mutagenesis by ran-dom cloning of antibiotic-resistance genes into the genome of the cya-nobacterium synechocystis strain Pcc-6803. J Bacteriol 171: 3449–3457

Lagarde D, Beuf L, Vermaas W (2000) Increased production of zeaxanthinand other pigments by application of genetic engineering techniques toSynechocystis sp. strain PCC 6803. Appl Environ Microbiol 66: 64–72

Lam MK, Lee KT (2012) Microalgae biofuels: a critical review of issues,problems and the way forward. Biotechnol Adv 30: 673–690

Landsberg JH (2010) The effects of harmful algal blooms on aquatic orga-nisms. Rev Fish Sci 10: 113–390

Lee AK, Lewis DM, Ashman PJ (2009) Microbial flocculation, a potentiallylow-cost harvesting technique for marine microalgae for the productionof biodiesel. J Appl Phycol 21: 559–567

Lengke MF, Ravel B, Fleet ME, Wanger G, Gordon RA, Southam G (2006)Mechanisms of gold bioaccumulation by filamentous cyanobacteriafrom gold(III)-chloride complex. Environ Sci Technol 40: 6304–6309

Levy N, Bar-Or Y, Magdassi S (1990) Flocculation of bentonite particles bya cyanobacterial bioflocculant. Colloids Surf 48: 337–349

Levy N, Magdassi S, Bar-Or Y (1992) Physico-chemical aspects in floccu-lation of bentonite suspensions by a cyanobacterial bioflocculant. WaterRes 26: 249–254

Liu G, Fan C, Zhong J, Zhang L, Ding S, Yan S, Han S (2010) Usinghexadecyl trimethyl ammonium bromide (CTAB) modified clays toclean the Microcystis aeruginosa blooms in Lake Taihu, China. HarmfulAlgae 9: 413–418

Liu X, Sheng J, Curtiss R III (2011) Fatty acid production in geneticallymodified cyanobacteria. Proc Natl Acad Sci USA 108: 6899–6904


Amezaga et al.


http://genome.microbedb.jp/cyanobase

http://genome.microbedb.jp/cyanobase


Ludwig M, Bryant DA (2012) Synechococcus sp. strain PCC 7002 tran-scriptome: acclimation to temperature, salinity, oxidative stress andmixotrophic conditions. Front Microbiol 3: 354

Maeso ES, Piñas FF, Gonzalez MG, Valiente EF (1987) Sodium require-ment for photosynthesis and its relationship with dinitrogen fixationand the external CO2 concentration in cyanobacteria. Plant Physiol 85:585–587

Magnusson MK, Koivisto Hursti UK (2002) Consumer attitudes towardsgenetically modified foods. Appetite 39: 9–24

Marin K, Kanesaki Y, Los DA, Murata N, Suzuki I, Hagemann M (2004)Gene expression profiling reflects physiological processes in salt accli-mation of Synechocystis sp. strain PCC 6803. Plant Physiol 136: 3290–3300

Markou G, Chatzipavlidis I, Georgakakis D (2012) Carbohydrates pro-duction and bio-flocculation characteristics in cultures of Arthrospira(Spirulina) platensis: improvements through phosphorus limitation pro-cess. Bioenerg Res 5: 915–925

Marks JS (2006) Taking the public seriously: the case of potable and nonpotable reuse. Desalination 187: 137–147

Marraccini P, Bulteau S, Cassier-Chauvat C, Mermet-Bouvier P, Chauvat F(1993) A conjugative plasmid vector for promoter analysis in severalcyanobacteria of the genera Synechococcus and Synechocystis. Plant Mol Biol23: 905–909

Matsuda N, Kobayashi H, Katoh H, Ogawa T, Futatsugi L, Nakamura T,Bakker EP, Uozumi N (2004) Na+-dependent K+ uptake Ktr system fromthe cyanobacterium Synechocystis sp. PCC 6803 and its role in the earlyphases of cell adaptation to hyperosmotic shock. J Biol Chem 279: 54952–54962

Mazouni K, Bulteau S, Cassier-Chauvat C, Chauvat F (1998) Promoterelement spacing controls basal expression and light inducibility of thecyanobacterial secA gene. Mol Microbiol 30: 1113–1122

McDaniel L, Houchin LA, Williamson SJ, Paul JH (2002) Plankton blooms:Lysogeny in marine Synechococcus. Nature 415: 496–496

Miller AG, Turpin DH, Canvin DT (1984) Na+ requirement for growth,photosynthesis, and pH regulation in the alkalotolerant cyanobacteriumSynechococcus leopoliensis. J Bacteriol 159: 100–106

Muhlenhoff U, Chauvat F (1996) Gene transfer and manipulation in thethermophilic cyanobacterium Synechococcus elongatus. Mol Gen Genet252: 93–100

Mukherjee J, Karunakaran E, Biggs CA (2012) Using a multi-faceted ap-proach to determine the changes in bacterial cell surface propertiesinfluenced by a biofilm lifestyle. Biofouling 28: 1–14

Muramatsu M, Hihara Y (2006) Characterization of high-light-responsivepromoters of the psaAB genes in Synechocystis sp. PCC 6803. Plant CellPhysiol 47: 878–890

Nakamura Y, Kaneko T, Tabata S (2000) CyanoBase, the genome databasefor Synechocystis sp. strain PCC6803: status for the year 2000. NucleicAcids Res 28: 72

Nie M, Yin X, Jia J, Wang Y, Liu S, Shen Q, Li P, Wang Z (2011) Pro-duction of a novel bioflocculant MNXY1 by Klebsiella pneumoniae strainNY1 and application in precipitation of cyanobacteria and municipalwastewater treatment. J Appl Microbiol 111: 547–558

O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbialdevelopment. Annu Rev Microbiol 54: 49–79

Olguín EJ (2012) Dual purpose microalgae-bacteria-based systems thattreat wastewater and produce biodiesel and chemical products within abiorefinery. Biotechnol Adv 30: 1031–1046

Pan G, Zhang MM, Chen H, Zou H, Yan H (2006a) Removal of cyano-bacterial blooms in Taihu Lake using local soils. I. Equilibrium andkinetic screening on the flocculation of Microcystis aeruginosa usingcommercially available clays and minerals. Environ Pollut 141: 195–200

Pan G, Zou H, Chen H, Yuan X (2006b) Removal of harmful cyanobacterialblooms in Taihu Lake using local soils. III. Factors affecting the removalefficiency and an in situ field experiment using chitosan-modified localsoils. Environ Pollut 141: 206–212

Peca L, Kós PB, Máté Z, Farsang A, Vass I (2008) Construction of biolu-minescent cyanobacterial reporter strains for detection of nickel, cobaltand zinc. FEMS Microbiol Lett 289: 258–264

Po M, Kaercher JD, Nancarrow BE (2003) Literature Review of FactorsInfluencing Public Perceptions of Water Reuse, CSIRO Land and WaterTechnical Report 54/03. Commonwealth Scientific and Industrial Re-search Organisation, South Clayton, Victoria, Australia

Porchia AC, Salerno GL (1996) Sucrose biosynthesis in a prokaryotic or-ganism: presence of two sucrose-phosphate synthases in Anabaena withremarkable differences compared with the plant enzymes. Proc NatlAcad Sci USA 93: 13600–13604

Porter RD (1986) Transformation in cyanobacteria. Crit Rev Microbiol 13:111–132

Prokop P, Ozel M, Usak M, Senay I (2013) Disease-threat model explainsacceptance of genetically modified products. Psihologija 46: 229–243

Pushparaj B, Pelosi E, Torzillo G, Materassi R (1993) Microbial biomassrecovery using a synthetic cationic polymer. Bioresour Technol 43: 59–62

Qi Q, Hao M, Ng WO, Slater SC, Baszis SR, Weiss JD, Valentin HE (2005)Application of the Synechococcus nirA promoter to establish an inducibleexpression system for engineering the Synechocystis tocopherol pathway.Appl Environ Microbiol 71: 5678–5684

Raghukumar C, Vipparty V, David JJ, Chandramohan D (2001) Degra-dation of crude oil by marine cyanobacteria. Appl Microbiol Biotechnol57: 433–436

Richardson DL, Reed RH, Stewart WDP (1983) Synechocystis PCC6803: aeuryhaline cyanobacterium. FEMS Microbiology Letters 18: 99–102

Schlesinger A, Eisenstadt D, Bar-Gil A, Carmely H, Einbinder S, GresselJ (2012) Inexpensive non-toxic flocculation of microalgae contradictstheories; overcoming a major hurdle to bulk algal production. Bio-technol Adv 30: 1023–1030

Schobert B, Lanyi JK (1982) Halorhodopsin is a light-driven chloridepump. J Biol Chem 257: 10306–10313

Seki A, Miyauchi S, Hayashi S, Kikukawa T, Kubo M, Demura M,Ganapathy V, Kamo N (2007) Heterologous expression of Pharaonishalorhodopsin in Xenopus laevis oocytes and electrophysiological char-acterization of its light-driven Cl- pump activity. Biophys J 92: 2559–2569

Shao J, Wang Z, Liu Y, Liu H, Peng L, Wei X, Lei M, Li R (2012) Physi-ological responses of Microcystis aeruginosa NIES-843 (cyanobacterium)under the stress of chitosan modified kaolinite (CMK) loading. Ecotox-icology 21: 698–704

Shibata M, Katoh H, Sonoda M, Ohkawa H, Shimoyama M, Fukuzawa H,Kaplan A, Ogawa T (2002) Genes essential to sodium-dependent bi-carbonate transport in cyanobacteria: function and phylogenetic analy-sis. J Biol Chem 277: 18658–18664

Siegrist M (2008) Factors influencing public acceptance of innovative foodtechnologies and products. Trends Food Sci Technol 19: 603–608

Sigalevich P, Meshorer E, Helman Y, Cohen Y (2000) Transition fromanaerobic to aerobic growth conditions for the sulfate-reducing bacte-rium Desulfovibrio oxyclinae results in flocculation. Appl Environ Mi-crobiol 66: 5005–5012

Silva PG, Silva HJ (2007) Effect of mineral nutrients on cell growth andself-flocculation of Tolypothrix tenuis for the production of a biofertilizer.Bioresour Technol 98: 607–611

Sims JH, Baumann DD (1974) Renovated waste water: the question ofpublic acceptance. Water Resour Res 10: 659–665

Simões LC, Simões M, Vieira MJ (2008) Intergeneric coaggregation amongdrinking water bacteria: evidence of a role for Acinetobacter calcoace-ticus as a bridging bacterium. Appl Environ Microbiol 74: 1259–1263

Singh NK, Dhar DW (2010) Cyanobacterial reclamation of salt-affectedsoil. In E Lichtfouse, ed, Genetic Engineering, Biofertilisation, SoilQuality and Organic Farming, Ed 1 Vol 4. Springer, Dijon, France, pp245–249

Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EPC, de la Cruz F(2010) Mobility of plasmids. Microbiol Mol Biol Rev 74: 434–452

Sorgo A, Jaušovec N, Jaušovec K, Pukek M (2012) The influence of intel-ligence and emotions on the acceptability of genetically modified or-ganisms. Electron J Biotechnol 15: 1

Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercialapplications of microalgae. J Biosci Bioeng 101: 87–96

Stevens SE, Porter RD (1980) Transformation in Agmenellum quad-ruplicatum. Proc Natl Acad Sci USA 77: 6052–6056

Su Y, Mennerich A, Urban B (2011) Municipal wastewater treatment andbiomass accumulation with a wastewater-born and settleable algal-bacterial culture. Water Res 45: 3351–3358

Sullivan MB, Waterbury JB, Chisholm SW (2003) Cyanophages infectingthe oceanic cyanobacterium Prochlorococcus. Nature 424: 1047–1051

Sun F, Pei HY, Hu WR, Li XQ, Ma CX, Pei RT (2013) The cell damage ofMicrocystis aeruginosa in PACl coagulation and floc storage processes.Sep Purif Technol 115: 123–128





Takaara T, Sano D, Konno H, Omura T (2007) Cellular proteins of Mi-crocystis aeruginosa inhibiting coagulation with polyaluminum chloride.Water Res 41: 1653–1658

Takaara T, Sano D, Masago Y, Omura T (2010) Surface-retained organicmatter of Microcystis aeruginosa inhibiting coagulation with poly-aluminum chloride in drinking water treatment. Water Res 44: 3781–3786

Takaichi S, Mochimaru M (2007) Carotenoids and carotenogenesis in cy-anobacteria: unique ketocarotenoids and carotenoid glycosides. CellMol Life Sci 64: 2607–2619

Tan X, Liang F, Cai K, Lu X (2013) Application of the FLP/FRT recombi-nation system in cyanobacteria for construction of markerless mutants.Appl Microbiol Biotechnol 97: 6373–6382

Taniguchi M, Kato K, Shimauchi A, Ping X, Nakayama H, Fujita KI,Tanaka T, Tarui Y, Hirasawa E (2005) Proposals for wastewater treat-ment by applying flocculating activity of cross-linked poly-g-glutamicacid. J Biosci Bioeng 99: 245–251

Teixeira MR, Sousa V, Rosa MJ (2010) Investigating dissolved air flotationperformance with cyanobacterial cells and filaments. Water Res 44: 3337–3344

Thajussin N, Subramanian G (2005) Cyanobacterial biodiversity and po-tential applications in biotechnology. Curr Sci 89: 47–57

Uduman N, Qi Y, Danquah MK, Hoadley AFA (2010) Marine microalgaeflocculation and focused beam reflectance measurement. Chem Eng J162: 935–940

United Nations (2006) Human Development Report 2006. Beyond Scarcity:Power, Poverty and the Global Water Crisis. Palgrave Macmillan, NewYork

Van Baalen C, Hoare DS, Brandt E (1971) Heterotrophic growth of blue-gren algae in dim light. J Bacteriol 105: 685–689

Verspagen JMH, Visser PM, Huisman J (2006) Aggregation with claycauses sedimentation of the buoyant cyanobacteria Microcystis spp.Aquat Microb Ecol 44: 165–175

Vioque A (2007) Transformation of cyanobacteria. Transgenic MicroalgaeAs Green Cell Factories 616: 12–22

Wang HL, Postier BL, Burnap RL (2004) Alterations in global patterns ofgene expression in Synechocystis sp. PCC 6803 in response to inorganiccarbon limitation and the inactivation of ndhR, a LysR family regulator.J Biol Chem 279: 5739–5751

Weigele PR, Pope WH, Pedulla ML, Houtz JM, Smith AL, Conway JF,King J, Hatfull GF, Lawrence JG, Hendrix RW (2007) Genomic and

structural analysis of Syn9, a cyanophage infecting marine Pro-chlorococcus and Synechococcus. Environ Microbiol 9: 1675–1695

WiangnonK, Raksajit W, Incharoensakdi A (2007) Presence of a Na+-stimulatedP-type ATPase in the plasma membrane of the alkaliphilic halotolerantcyanobacterium Aphanothece halophytica. FEMS Microbiol Lett 270: 139–145

Wilde A, Dienst D (2011) Tools for Genetic Manipulation of Cyanobacteria.In GA Peschek, C Obinger, G Renger, eds, Bioenergetic Processes ofCyanobacteria: From Evolutionary Singularity to Ecological Diversity,Springer, New York, pp 685–703

World Health Organization (2007) Desalination for Safe Water Supply:Guidance for the Health and Environmental Aspects Applicable to De-salination. Geneva, Switzerland, World Health Organization

Xu Y, Alvey RM, Byrne PO, Graham JE, Shen G, Bryant DA (2011) Ex-pression of genes in cyanobacteria: adaptation of endogenous plasmidsas platforms for high-level gene expression in Synechococcus sp. PCC7002. Methods Mol Biol 684: 273–293

Xue S, Yao X, Luo W, Jha D, Tester M, Horie T, Schroeder JI (2011)AtHKT1;1 mediates nernstian sodium channel transport properties inArabidopsis root stelar cells. PLoS ONE 6: e24725

Yoshihara S, Geng X, Ikeuchi M (2002) pilG Gene cluster and split pilLgenes involved in pilus biogenesis, motility and genetic transformationin the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 43:513–521

Yoshihara S, Geng XX, Okamoto S, Yura K, Murata T, Go M, Ohmori M,Ikeuchi M (2001) Mutational analysis of genes involved in pilus struc-ture, motility and transformation competency in the unicellular motilecyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 42: 63–73

Zeleznik MJ, Segatta JM, Ju LK (2002) Polyethyleneimine-induced floc-culation and flotation of cyanobacterium Anabaena flos-aquae for gasvesicle production. Enzyme Microb Technol 31: 949–953

Zang XN, Liu B, Liu SM, Arunakumara KKIU, Zhang XC (2007) Optimumconditions for transformation of Synechocystis sp PCC 6803. J Microbiol45: 241–245

Zhang F, Vierock J, Yizhar O, Fenno LE, Tsunoda S, Kianianmomeni A,Prigge M, Berndt A, Cushman J, Polle J, et al (2011) The microbialopsin family of optogenetic tools. Cell 147: 1446–1457

Zou H, Pan G, Chen H, Yuan X (2006) Removal of cyanobacterial blooms inTaihu Lake using local soils. II. Effective removal of Microcystis aerugi-nosa using local soils and sediments modified by chitosan. EnvironPollut 141: 201–205


Amezaga et al.



Biodesalination: A Case Study for Applications of...Biodesalination: A Case Study for Applications...

Documents

Transcript of Biodesalination: A Case Study for Applications of...Biodesalination: A Case Study for Applications...