Plant Physiology Preview. Published on March 7, 2014, as DOI:10.1104/pp.113.233973
‘Biodesalination’: a case study for applications of photosynthetic
bacteria in water treatment
Jaime M. Amezaga1, Anna Amtmann2*, Catherine A. Biggs3, Tom Bond4, Catherine J. Gandy1,
Annegret Honsbein2, Esther Karunakaran3, Linda Lawton5, Mary Ann Madsen2, Konstantinos
Minas5, Michael R. Templeton4
1
School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne, UK
NE1 7RU, UK
2
Institute of Molecular, Cell and Systems Biology, MVLS, University of Glasgow, Glasgow, G12
8QQ, UK
3
Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD,
UK
4
Department of Civil and Environmental Engineering, Imperial College London, London SW7
2AZ, UK
5
Institute for Innovation, Design and Sustainability, Robert Gordon University, Aberdeen
*Corresponding author: [email protected]
Abstract
Shortage of freshwater is a serious problem in many regions worldwide, and expected to become
even more urgent over the next decades due to increased demand for food production and adverse
effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable,
energy-efficient technologies for desalination are developed. Energization of desalination by
sunlight through photosynthetic organisms offers a potential opportunity to exploit biological
processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in
brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could
be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In
this review we use the example of ‘biodesalination’ as a vehicle to review the availability of tools
and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to
strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation,
process design, safety and public acceptance.
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Copyright 2014 by the American Society of Plant Biologists
Bacteria are commonly employed for the purification of municipal and industrial wastewater, but
until now established water treatment technologies have not taken advantage of photosynthetic
bacteria i.e. cyanobacteria. The ability of cyanobacterial cultures to grow at high cell densities with
minimal nutritional requirements – sunlight, carbon dioxide and minerals - opens up many future
avenues for sustainable water treatment applications.
Water security is an urgent global issue, especially since many regions of the world are
experiencing, or are predicted to experience, water shortage conditions: over one in six people
globally are water-stressed, in that they do not have access to safe drinking water (UN, 2006). 97%
of the Earth’s water is in the oceans and consequently there are many efforts to develop efficient
methods for converting saltwater into freshwater. Various processes using synthetic membranes,
such as reverse osmosis, are successfully used for large scale desalination, however, the high energy
consumption of these technologies have limited their application predominantly to countries with
both relatively limited freshwater resources and high availability of energy, for example, in the form
of oil reserves.
Therefore, the development of an innovative, low-energy biological desalination process,
using biological membranes of cyanobacteria, would be both attractive and pertinent. The core of
the proposed ‘biodesalination’ process (Figure 1) is a low-salt biological reservoir within seawater
that can serve as an ion exchanger. Its development can be separated into several complementary
steps: 1. Selection of a cyanobacterial strain which can be grown to high cell-densities in seawater
with minimal requirement for energy sources other than those naturally available. The
environmental conditions during growth can be manipulated to enhance natural extrusion of sodium
(Na+) by cyanobacteria. 2. Manipulation of cyanobacterial ion transport mechanisms, to generate
cells in which sodium export is replaced with intracellular sodium accumulation This will involve
inhibition of endogenous Na+ export and expression of synthetic molecular units that facilitate lightdriven sodium flux into the cells. A robust control system built from biological switches will be
required to achieve precisely timed expression of the salt accumulating molecular units. 3.
Engineering efficient separation of the cyanobacterial cells from the desalinated water, using
knowledge of physicochemical properties of the cell surface and their natural ability to produce
extracellular polymeric substances (EPS), which aid cell separation whilst preserving cell integrity.
4. Integration of steps 1 to 3 into a manageable and scalable engineering process. 5. Assessment of
potential risks and public acceptance issues linked to the new technology.
In this review we will outline the state of knowledge and available technology for each of
the steps, as well as summarize the current knowledge gaps and technical limitations in employing a
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large scale water treatment process using cyanobacteria. Before discussing these issues, we provide
some background information on usage of cyanobacteria in biotechnology and the impact of sodium
on cellular functions of cyanobacteria. The example of biodesalination provides a good vehicle to
discuss the suitability of photosynthetic bacteria for water treatment more generally. The issues
addressed in this review are relevant for a wide range of biotechnological applications of
cyanobacteria, including bioremediation and biodegradation, as well as the generation of biofuel,
natural medicines or cosmetics.
CYANOBACTERIA IN BIOTECHNOLOGY
Cyanobacteria are a phylum of photosynthetic, oxygen-producing bacteria, with a long
evolutionary history (Altermanna and Kazmierczak, 2003). Due to the process of “complementary
chromatic adaptation” (Bennett and Bogorad, 1973), cyanobacteria can utilize a wide spectrum of
photosynthetically active radiation (PAR) as their primary source of energy. The long evolutionary
history has allowed them to adapt to a wide range of environmental conditions and to occupy a vast
array of ecological niches. Modest growth requirements combined with high adaptability generate a
potential for harmful algal blooms, which have earned these organisms some bad publicity.
However, cyanobacteria have contributed to human nutrition for millennia either directly as a food
source or indirectly through nitrogen fixation in rice paddies (Landsberg, 2010; Thajussin and
Subramanian, 2005). More recently, biotechnological applications of cyanobacteria have allowed
for their utilization as animal feeds and human food supplementation, and as producers of
bioenergy, cosmetics, anti-cancer and anti-HIV drugs (Spolaore et al. 2006).
In the context of environmental cleanup, Oscillatoria salina, Plectonema terebrans and
Aphatnocapsa sp. have been used successfully for the degradation of crude oil in seawater
(Raghukumar et al. 2001). The applicability of cyanobacteria extends to the remediation of heavy
metals, e.g. cadmium by Tolypothric tenuis, or even the reclamation of precious metals, e.g. gold by
Plectonema boryanum (Inthorn et al. 1996; Lenkge et al. 2006). An indication for possible usage of
cyanobacteria for desalination was the reclamation of saline soils in India and the USSR using
endemic strains (Apte and Thomas, 1997; Singh and Dhar, 2010). Thus, removal of cyanobacterial
mats formed after rainfall also removed salt from the soil. Further investigation of Anabaena
torulosa (a brackish strain) and Anabaena L-31 (a freshwater strain) demonstrated that 90% of the
salt accumulated was bound to extracellular polymeric substances (EPS) at the cell surface, while
the remainder was internalized and osmotically active. The freshwater strain showed a higher net
sodium uptake than the brackish strain, probably due to higher sodium efflux capacity of the latter.
Interestingly, the influx of sodium was diminished in both strains by alkaline pH, high amount of
extracellular potassium, or the presence of nitrates or ammonium (Apte and Thomas, 1983; Apte et
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al. 1987). These observations suggested that environmental triggers could be used to alter the
magnitudes of sodium influx and efflux through endogenous transport systems.
SELECTION OF SUITABLE CYANOBACTERIAL STRAINS
Strain selection for biotechnological applications needs to be guided by the purpose and the
environment of the envisaged process. With respect to biodesalination, candidate strains should
meet a few key criteria. The culture should be fast-growing, to allow for the generation of high cell
density within short time, thereby generating a large cumulative internal volume and a large total
cell surface. The strain should be able to grow over a wide range of external salt concentrations; the
cells should be able to adjust osmotically and to effectively export Na+ during growth. To allow for
cell separation from the water and other post-treatment procedures the cells should preferably be
unicellular, possess a cell wall and EPS, and have the capacity to adjust their buoyancy, e.g. through
intracellular gas vesicles. Finally, to facilitate genetic manipulation, the cells should be amenable to
transformation techniques and their genome sequence should be known.
Based on these criteria and an initial screen carried out in one of our laboratories (Figure 2),
two strains
emerge as attractive candidates for biodesalination: the fresh water-euryhaline
Synechocystis sp. PCC 6803 strain (Richardson et al., 1983) and the marine-euryhaline
Synechococcus sp. strain PCC 7002 (formally Agmenellum quadruplicatum PR-6) (Ludwig and
Bryant, 2012). Both strains are unicellular, capable of axenic growth and easy to maintain under
laboratory conditions. The genomes of both organisms have been sequenced (KDRI, 2013) and
successful transformation with foreign DNA has been reported (see below). A particular advantage
of Synechococcus PCC 7002 is its high growth rate. Generation times of less than three hours have
been reported, making this strain the fastest dividing cyanobacterium and one of the fastest growing
photosynthetic organisms (Van Baalen et al, 1971). Both strains have been used extensively as
models for the study of photosynthesis. This research has already provided a wealth of scientific
knowledge including information on physiological adaptations to salinity and other environmental
factors (Nakamura et al, 2000; Ludwig and Bryant, 2012).
MANIPULATION OF ENDOGENOUS SODIUM TRANSPORT IN CYANOBACTERIA
Any usage of unicellular systems such as cyanobacteria for the removal of sodium (Na+)
from seawater or brackish water requires an understanding of the potential effects of Na+ on cellular
functions, which in turn depend on the Na+-concentration. Some Na+ is necessary for nutrient
uptake (e.g. Na+-dependent HCO3- transport), nitrate assimilation, nitrogen fixation and
photosynthesis (Apte and Thomas 1983; Maeso et al, 1987; Espie et al, 1988). Na+ is also required
for cell division in heterotrophic cyanobacteria, and for pH homeostasis in alkaline environments
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(Miller et al. 1984). When the intracellular sodium concentration exceeds a certain level deleterious
effects become apparent including destabilization of the fatty acids in the cell membrane (Huflejt et
al, 1990), inhibition of electron transport between H2O and photosystem II (Allakhverdiev and
Murata, 2008) and a complete halt of photoautotrophic growth (Bhargava et al, 2003). The exact
level at which Na+ becomes toxic depends on both endogenous and environmental factors and
differs between strains.
Successful salt acclimation of cyanobacterial cells depends on ambient concentrations and
length of the exposure (Marin et al. 2004; Hagemann, 2010). It is a multistage process that includes
the re-adjustment of ionic and osmotic potentials as well as wider physiological changes. Turgor
adjustment is one of the earliest responses to salt stress (Blumwald et al, 1983). It involves the
biogenesis and accumulation of compatible solutes such as glycosylglycerol (GG) and sucrose
(Porchia and Salerno, 1996; Engelbrecht et al, 1999). Moreover, increased osmolyte uptake has
been observed in some strains under salt stress, and this uptake appeared to alleviate some of the
effects caused by salinity (Fulda et al, 1999). If salt stress persists, ionic adjustment becomes
increasingly important, in particular the active extrusion of Na+ through Na+/H+ antiporters, as well
as P-type Na+-ATPases (Marin et al, 2004; Wiagnon et al. 2007).
Environmental manipulations can make use of factors that directly or indirectly alter the
metabolism of the organism. The primary metabolism of cyanobacteria is largely based on
photosynthesis and hence strongly regulated by light. By altering the photoperiod, light intensity or
wavelength, metabolic processes can be induced or inhibited literally by the “flick of a switch”.
Availability of carbon, nitrate and phosphate also exert significant control over growth, metabolism
and energy status. In particular, co-transport of bicarbonate, phosphate and nitrate with Na+ (Shibata
et al, 2002; Matsuda et al, 2004, Baebprasert et al, 2011) opens opportunities to use these
macronutrients to modulate Na+ uptake rates. Altering the cell’s energy status through metal
deficiencies will impact on active Na+-export from the cell, which consumes a large proportion of
the cell’s ATP. Magnesium in chlorophyll and iron in heme groups are essential components of the
photosystem and hence required for photosynthetic activity while inorganic phosphate is required
for oxidative phosphorylation. Deficiency of these elements is the most common reason for cultures
entering stationary phase and it can therefore be expected that cells lose their capacity to exclude
Na+ towards the end of the growth period. Furthermore, metabolic activity is affected by changes in
pH and temperature. A systematic assessment of the effects of individual factors, and of their
combinations, on Na+ transport in Synechococcus PCC 7002 and Synechocystis PCC 6803 is now
required to provide a set of environmental triggers that can be used to alter Na+ exchange between
the cells and the surrounding water.
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GENETIC MANIPULATION OF CYANOBACTERIA
The two methods that are most commonly used for transferring foreign genetic material into
cyanobacteria are natural transformation and conjugation. Several detailed reviews have been
published on the genetic manipulation of cyanobacteria in general (Heidorn et al., 2011;
Koksharova and Wolk, 2002; Vioque, 2007; Wilde and Dienst, 2011). Here we will only give a
short overview with emphasis on available tools for Synechococcus PCC 7002 and Synechocystis
PCC 6803.
Natural transformation
Natural transformation involves the spontaneous uptake of DNA from the environment and
subsequent integration into the host genome. Both Synechococcus PCC 7002 (Essich et al., 1990;
Frigaard et al., 2004; Stevens and Porter, 1980) and Synechocystis PCC 6803 (Barten and Lill,
1995; Grigorieva and Shestakov, 1982; Heidorn et al., 2011) are naturally transformable, although
the process of DNA uptake is incompletely understood. Amongst other factors, type IV pili, which
are also responsible for cell mobility, are an important part of the natural competence of
Synechocystis PCC 6803 (Yoshihara et al., 2001; Yoshihara et al., 2002). Only double-stranded
DNA can be used for natural transformation, but it is converted into single-stranded DNA as it
passes through the cell envelope. Inside the cell the double-stranded state is restored during
recombination with the chromosomal or plasmid DNA of the host (Barten and Lill, 1995; Essich et
al., 1990). A calcium-dependent nuclease, located in or on the plasma membrane, was proposed to
be responsible for the degradation of one of the two strands during DNA uptake in Synechocystis
PCC 6803 (Barten and Lill, 1995). In PCC 6803, no further fragmentation of extracellular DNA
incorporated into the cell in this manner was observed (Barten and Lill, 1995; Kufryk et al., 2002).
So-called integrative or suicide plasmids are used for natural transformation in the
laboratory. These plasmids are able to replicate in E. coli, which is used for cloning of the gene
before transfer to the cyanobacterial host. They allow the researcher to position the gene of interest
between two flanking regions of DNA that are homologous to sequences of the cyanobacterial
genome, the so called 'neutral sites'. Neutral sites are regions whose deletion or interruption has
produced no phenotypic effect under all growth conditions investigated so far. Neutral sites are
generally found in silent or redundant genes as opposed to intergenic or 3′-untranslated regions,
which can execute regulatory functions on gene expression (Wilde and Dienst, 2011). In
Synechocystis PCC 6803 and Synechococcus PCC 7002 integration of foreign DNA between the
two flanking homologous regions usually occurs by a double crossover event mediated by the
highly efficient homologous recombination system of these strains. The recombination efficiency
depends on the length of the homologous stretches. The optimal length is different for different
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strains but generally the longer the better (Heidorn et al., 2011; Labarre et al., 1989; Xu et al.,
2011). Covalently closed or linearized plasmids as well as PCR products of the region of interest
can be used in natural transformation. For Synechocystis PCC 6803 transformation with circular
plasmid DNA was found to be approximately 30% more efficient than transformation with
linearized plasmid DNA (Kufryk et al., 2002), while for Synechococcus PCC 7002 the use of linear
fragments was recommended to achieve high transformation efficiency (Xu et al., 2011).
Conjugation
DNA transfer by conjugation consists of plasmid exchange between different bacteria. In the
laboratory, three strains are typically used for conjugation, also called tri-parental mating: the host
cyanobacterium and two E. coli strains. One E. coli strain carries the vector containing the gene of
interest (cargo plasmid) and the second E. coli strain carries the conjugal plasmid. If additional
helper plasmids are needed they usually join the cargo plasmid in the first E. coli strain. Mixing of
the two E. coli strains causes the conjugative plasmid to transfer to the E. coli strain that carries the
cargo and helper plasmids. The latter is then competent to conjugate with the subsequently added
cyanobacterial strain and to transfer the cargo plasmid to the new host (Vioque, 2007; Wilde and
Dienst, 2011).
The cargo plasmids used for conjugation are vectors capable of autonomous replication in
the host cyanobacterium, as well as in E .coli, where the initial cloning takes places. Two types of
vectors can be distinguished. Shuttle vectors are hybrids between a native cyanobacterial plasmid
and an E. coli plasmid and therefore carry two different origins of replication, one that is specific
for the particular cyanobacterium and one that is specific for E. coli. Broad host range vectors carry
only one replicon, which functions in many different bacterial hosts including cyanobacteria and E.
coli (Heidorn et al., 2011).
For conjugal transfer of both types of vectors, certain additional genetic elements are
essential. Most importantly, in the donor cell, a relaxase/nickase of the mobility gene family (mob
genes) recognizes and cleaves a specific site within an origin of transfer (oriT). The DNA strand
with the covalently bound relaxase protein is displaced from the plasmid by an ongoing conjugative
DNA replication process. Through interaction of the relaxase with components of a multiprotein,
membrane-associated mating pair formation complex, a type IV secretion system (tra genes), it is
transported to the recipient cell together with the attached DNA. In the recipient cell the relaxase
catalyzes the ligation of the transported DNA to reconstitute the conjugated plasmid (Smillie et al.,
2010). The oriT is the only sequence required in cis for a plasmid to be conjugally transmissible,
which is why both the shuttle vector and the broad host vectors carry this DNA sequence.
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Other techniques for DNA transfer
Protocols enabling DNA transfer through electroporation have been developed for Synechocystis
PCC 6803 (Marraccini et al., 1993; Zang et al., 2007), but cell recovery after the procedure is slow
and there are reports that this technique increases mutation rates in some cyanobacteria (Bruns et
al., 1989; Muhlenhoff and Chauvat, 1996). In the future, transfer of DNA through cyanobacteral
viruses (cyanophages) could become an attractive alternative although appropriate genetic tools for
transduction have not yet been published. However, non-lytic cyanophages that infect marine
Synechococcus species and have their genome stably maintained within thehost have already been
described (McDaniel et al., 2002). Furthermore, it is known that some cyanophages have a broad
host range and can cross-infect both Prochlorococcus and closely related Synechococcus species,
which has been implicated in horizontal gene transfer of photosynthesis-related genes (Sullivan et
al., 2003; Weigele et al., 2007). Those types of phages have potential for the development of
genetic tools.
Technique of choice and current limitations
The technique of choice for the genetic manipulation of Synechocystis PCC 6803 and
Synechococcus PCC 7002 will depend on how the foreign gene information should be maintained in
the host cyanobacterium. As mentioned above, the process of natural transformation involves DNA
linearization and conversion to a single strand (Porter, 1986), which makes this technique
unsuitable for genes on an autonomously replicating plasmid. In this case conjugation is the
method of choice as it ensures that at the end of the transfer a circular plasmid resides in the host
(Vioque, 2007). Integration into the host genome by natural transformation is desirable when longterm inheritance is the goal. It also potentially reduces gene dose variation caused by copy number
variations of autonomously replicating plasmids. The downside of incorporation of foreign DNA by
homologous recombination into the genome is that cyanobacteria generally have multiple copies of
the chromosome (e.g. 12 in Synechocystis PCC 6803), and therefore heterozygous cells are created.
Subsequent segregation over several generations is needed to ensure that the foreign DNA is present
in all copies (Heidorn et al., 2011).
For applications beyond a laboratory setting, it is essential that marker genes (e.g. antibiotic
resistance genes) do not remain in the genome. To achieve marker-free genomic mutations counterselection procedures have been developed for both PCC 6803 and PCC 7002. For PCC 6803 the
process requires two transformation steps (Cheah et al.2013). With the first transformation, a
cassette containing two marker genes a kanamycin resistance gene for positive selection and the
mazF gene for negative selectionn is inserted into the genome via homologous recombination. mazF
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encodes a toxic endoribonuclease and is under the control of a nickel-inducible promoter.
Successfully transformed cells are selected on nickel-free kanamycin-containing media and
subjected to a second round of transformation, where the entire cassette inserted by the first
transformation is replaced with the gene of interest. Subsequent counter-selection is performed on
kanamycin-free, nickel-containing medium. Cells that have not lost the marker gene cannot grow
due to induction of mazF. A similar counter-selection method for PCC 6803 uses the B.subtilis
levan sucrase gene sacB as negative selection marker (Lagarde et al.2000). The disadvantage of this
counter-selection system is the requirement for a separate glucose-tolerant strain of PCC 6803 as
chassis. An alternative strategy for PCC 6803 is based on the recombinase-system FLP/FRT from
Saccharomyces cerevisiae rather than counter-selection. As with the above mentioned counterselection methods two transformation steps are needed (Tan et al.2013). A successful counterselection procedure for PCC 7002 is based on acrylate-toxicity and requires only one transformation
step (Begemann et al.2013). Deletion of the gene acsA through its replacement with the DNA
fragment of interest via homologous recombination overcomes growth inhibition by acrylate. Thus,
positive transformants are identified by their ability to grow on selective medium containing
acrylate. To achieve expression of multiple heterologous genes, the acsA gene can be re-inserted
into the genome at a neutral site, e.g. the pseudogene glpK. The organic acid counter-selection
method is potentially applicable also to PCC 6803, as AcsA activity confers acrylate sensitivity also
to this strain.
A major problem for the genetic manipulation of cyanobacteria consists in their efficient
system of restriction enzymes that destroy foreign DNA introduced by any transformation
technique. One way to prevent DNA fragmentation is to ensure that the introduced DNA sequence
contains no sites that are recognized by the endogenous restriction system. However, target sites
differ between cyanobacterial species, which is one reason why a shuttle or broad host vector that is
maintained in one species might be digested in another. A second approach is used in conjugation,
where the helper plasmid can encode methylases that protect against restriction enzymes commonly
present in many cyanobacteria (Vioque, 2007).
In conclusion, methods for genetic manipulation of cyanobacteria have been established but
the number of available tools is still limited. For example, a set of two integrative vectors exist for
PCC 7002 that recombine not with the chromosome but with endogenous plasmids (Xu et al.,
2011). As those can reach copy numbers of up to 50, high-level gene expression is achieved. This
elegant solution is not available for PCC 6803 yet. On the other hand, autonomously replicating
plasmids are still missing for PCC 7002, although a recently developed broad-host range vector is a
potential candidate (Huang et al., 2010).
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DESIGNING A SYNTHETIC BIODESALINTOR
Generation of a salt-free biological reservoir
The core of the proposed biodesalination process consists in the establishment of a salt-free (or lowsalt) biological reservoir within seawater that can serve as an ion exchanger. Most marine
organisms already contain such a reservoir because they actively exclude and remove salt from their
bodies. Cyanobacteria employ a range of Na+ export proteins in their cell membrane (Figure 3), all
of which are energized by the chemical energy carrier adenosine-tri-phosphate (ATP). ATP powers
Na+ export either directly through Na+-pumping ATPases, or indirectly through H+-pumping
ATPases, which generate a proton motive force that drives H+/Na+-antiport (Marin et al, 2004;
Wiagnon et al. 2007). The ATP-requirement offers an opportunity to halt Na+ export by depleting
internal ATP stores using the environmental manipulations detailed above, e.g. omitting
photosynthetically efficient wavelengths from the light spectrum, depleting phosphate, chelating
Mg2+, Fe2+ or other essential metals, altering pH etc. Simply changing the growth system from an
open system to a closed system once the culture has achieved high cell-density may already rapidly
deplete nutrient supply and exhaust ATP reserves.
Designing light-powered transport modules
Once active Na+ export has come to a standstill there will be net Na+ influx into a cell until
equilibrium with the external medium is reached. Further extraction of Na+ from the medium will
then require an energy source. To prevent renewal of Na+ export, the energy-harvesting system
employed during this phase should not use ATP as an intermediate. Good candidates for ATPindependent light-powered biological batteries are halorhodopsins (Hrs). Naturally occurring in
extremely salt-tolerant archaea (haloarchaea), Hrs are membrane-integral proteins of the rhodopsin
superfamily that form a covalent bond with the carotenoid-derived chromophore all-trans retinal
(Schobert and Lanyi 1982; Klare et al. 2008). Absorption of a photon with a defined optimal
wavelength induces trans-cis isomerization of retinal which triggers a catalytic photocycle of
conformational changes in the protein, resulting in the net import of one chloride per photon into
the cytoplasm. The turnover rates for light activated ion pumps such as Hr are in the millisecond
range (Kolbe et al. 2000; Kouyama et al. 2010; Essen 2002; Chizhov and Engelhard 2001).
To date, several Hr proteins from different species have been characterized (Klare et al.
2008; Fu et al. 2012). The Hr from Natronomonas pharaonis (NpHr) has been cloned and
successfully expressed in heterologous systems such as E.coli, mammalian cells and Xenopus laevis
oocytes (Hohenfeld et al.1999; Seki et al.2007; Gradinaru et al.2008). Expression of NpHr in
Xenopus laevis oocytes resulted in a light-dependent Cl--inward current and consequently
a
negative shift in the membrane potential (Seki et al.2007). The opportunity to artificially manipulate
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a cell’s membrane potential through NpHr in conjunction with light-activated cation channels (e.g.
channel rhodopsin) has been exploited in the field of optogenetics achieving control of action
potentials in nerve cells with potential medicinal applications (Fenno et al. 2011; Zhang et al.
2011). We propose here that the negative membrane potential generated by Hr could also be used
to drive the accumulation of positively charged substances in cells. Thus expression of Hr could
energize the uptake of nutrients (e.g. Ca2+, Mg2+, K+, Fe2+), or toxic metals (e.g. Cd2+, Ni2+), into
either plants or microorganisms, for biofortification and bioremediation, respectively.
Expression of Hr in a high-density cyanobacterial culture should remove both Cl- and Na+
from surrounding seawater and therefore provide a means for biodesalination. The observed K m
values of Hrs for chloride uptake (around 25 mM for chloride, Duschl et al. 1990) are in an optimal
range for this purpose. To increase the speed of Na+ accumulation, the Na+ conductance of the
membrane might need to be enhanced by co-expression of Na+-permeable channels or carriers with
Hr (Figure 3). Candidate proteins with different affinities and gating characteristics can be found in
bacteria (Koishi et al. 2004), animals (Koopmann et al. 2006) and plants (Xue et al.2011). The
resulting light-powered ‘salt-accumulator’ bypasses the endogenous energy metabolism
(photosynthesis and respiration) and should therefore remain functional even when increasing
intracellular Na+-levels inhibit other metabolic functions of the host. A living cell would thus be
transformed into a ‘synthetic’ cell.
Ensuring function and robustness of the synthetic biodesalinator
While technologies for environmental and genetic manipulation of cyanobacteria are advancing fast
and are predicted to enable realization of the core synthetic salt-accumulator, several additional
challenges remain to be solved. Firstly, only the protein part of halorhodopsin can be heterologously
expressed in cyanobacteria. The essential all-trans retinal is usually added as a supplement in the
laboratory, but this is not sustainable in a large scale process. Little is known about whether the
enzymes that produce all-trans retinal from β-carotene are present in cyanobacteria. However,
cyanobacteria as photosynthetic organisms already produce a wealth of carotenoids for light
harvesting and photo-protection (Takaichi and Mochimaru 2007) and thus engineering a synthetic
pathway for the final enzymatic steps should not prove too difficult. Secondly, progressive
accumulation of NaCl in the cells requires not only rapid osmotic adjustment of the cells (which
most cyanobacteria are capable of) but also threatens to lead to destabilization of membranes and
proteins. It is therefore important that the cyanobacterial strain is resistant to high salt
concentrations and that the heterologously expressed halorhodopsin and channel proteins are
derived from naturally salt-tolerant species. Additional measures such as increasing the external
Ca2+ concentration and altering lipid composition of the membrane should also be explored. Finally,
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even if the biological materials are salt-resistant the biodesalination process will need to be limited
to a very narrow time window situated between the end of the growth phase and the cell-removal
phase. It is therefore essential to obtain control over the expression of introduced genes.
Gaining control over gene expression in cyanobacteria
Control over gene expression is exerted through promoter regions in the DNA, usually located
immediately upstream of the gene, which are recognized by effectors (initiating transcription) as
well as other regulatory proteins that link transcriptional activity to endogenous and environmental
stimuli. Obtaining control over transgene expression in cyanobacteria requires the identification and
isolation of promoters that are responsive to the specific triggers that will be used in the
biotechnological process (environmental changes or supplements). For example, in the envisaged
biodesalination process, promoters that are specifically active in the early stationary phase of the
culture could be cloned into the expression vectors to activate the transgenes after the initial growth
period. To ensure specificity and precise timing of gene transcription the suitability of any
candidate promoter as ‘biological switch’ needs to be tested in a range of conditions and systems.
Promoter studies in cyanobacteria to date have primarily focused on characterizing native
transcriptional regulation in response to different environmental stimuli. Traditionally, PCC 6803
was studied as a model for photosynthesis and circadian rhythm and several light-responsive
promoters were identified, including the light-inducible LR-1 (Marraccini et al., 1993) and secA
(Mazouni et al., 1998), light-repressible psaAB (Muramatsu and Hihara, 2006) and dark-inducible
lrtA (Imamura et al., 2004) promoters. More recently, cyanobacterial studies have turned their focus
to biotechnological applications and numerous heavy metal-inducible promoters have been
characterized (Peca et al., 2008; Blasi et al., 2012) as well as the copper-inducible petE (Briggs et
al., 1990; Ghassemian et al., 1994; Buikema and Haselkorn, 2001) and copper-repressible petJ
(Ghassemian et al., 1994) promoters. Furthermore, promoters tightly regulated by nutrient
availability have been characterized including the sbtA promoter regulated by inorganic carbon
availability (Wang et al., 2004) and the nirA promoter regulated by nitrogen source (Ivanikova et
al., 2005; Qi et al., 2005).
The majority of studies characterizing cyanobacterial promoter activity have been performed
in the native organisms. This poses a problem for transgenic applications due to potential crosstalk
and/or recombination; therefore in biotechnology native promoters are generally avoided in favor of
promoters from closely-related organisms. The most common method of gene regulation in bacteria
is the lacI-lacO repression system, however, while this works well in some strains of cyanobacteria
such as Synechococcus sp. PCC 7942 (Clerico et al., 2007) it is not suitable for others including
PCC 6803 (Huang et al., 2010). Other promoters that are well characterized in E. coli such as the PL
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and PR promoters of bacteriophage lambda have also shown poor functionality in cyanobacteria
(Huang et al., 2010; Huang and Lindblad, 2013).
A range of different vectors and reporters have been used to test promoter activity in
cyanobacteria (Marraccini et al., 1993; Ivanikova et al., 2005; Peca et al., 2008; Huang et al., 2010;
Xu et al., 2011; Blasi et al., 2012). In an attempt to standardize the characterization of promoter
activity for synthetic biology applications, a method was developed in E. coli whereby promoter
activity could be measured relative to an in vivo reference promoter based on the fluorescence
intensities of green fluorescent protein (GFP) as a reporter (Kelly et al., 2009). The method was
further developed using a broad-host-range vector derived from the IncQ plasmid, RSF1010, for
promoter analysis in PCC 6803 (Huang et al., 2010). Due to the nature of the vector, this method
can be applied to a wide range of organisms likely to include other cyanobacterial species.
In summary, some promoters regulated by different stimuli s have been identified and
characterized in cyanobacteria. For these to be suitable for biotechnological applications, the
activity of these promoters must be characterized in non-native settings, and standardized methods
for characterization in cyanobacteria have been developed. At this stage the availability of effective
‘biological switches’ is still a bottleneck for usage of cyanobacteria as chassis in synthetic biology
and for biotechnological applications.
STRATEGIES FOR CELL-WATER SEPARATION
Once biodesalination has occurred, efficient cell-water separation is the next step of the proposed
process (Figure 1). The notion of
microorganisms as independent unicellular entities is
continuously challenged by research into microbial biofilms (O’Toole et al 2000). Nevertheless
exploitation of photosynthetic microorganisms in water treatment has focused predominantly on the
use of unicellular microbial suspensions, i.e. planktonic cells or suspended multispecies microflocs.
Whilst the ease of growth and maintenance favor use of planktonic cultures of cyanobacteria for
biodesalination,
the separation of such cells from the desalinated water during downstream
processing without affecting the integrity of the cells and inadvertent release of sodium chloride
back into the desalinated water, will be an economical and technical bottleneck in the
biodesalination process, as seen from previous attempts at water treatment using photosynthetic
microorganisms (Schlesinger et al 2012, Lam and Lee 2012, Olguin et al 2012, Uduman et al 2010).
The difficulty in separating planktonic cyanobacterial cells from aqueous suspensions stems from
the fact that cells have similar densities to water, behave like colloidal particles due to the
dimensions of the cells (few microns) and possess charged surfaces that stabilize cell suspensions.
Metal salts for coagulation
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The removal of photosynthetic microorganisms, especially bloom-forming cyanobacterial strains
such as Microcystis aeruginosa and Nodularia sp., from water has been studied in the context of
water treatment processes. Therefore the cell-liquid separation techniques have borrowed heavily
from wastewater treatment procedures although centrifugation and filtration are employed when
product quality, especially of high value chemicals, is to be ensured. Nevertheless, coagulation and
flocculation based processes are considered to be more energy efficient and cost effective than
centrifugation and filtration (Uduman et al 2010; Lam and Lee, 2012). Inorganic metal salts such as
aluminium sulphate (4.8mg/l – 5.8mg/l - Chow et al 1999; 65 mg/l - 70mg/l – Drikas et al 2001),
ferric chloride (30mg/l – Chow et al 1998) and poly aluminium chloride (4 mg/l - Sun et al 2013)
are effective at separating out up to 99% of cyanobacteria from water. Aggregation of cells with
addition of metal salts is mediated by the neutralisation of surface charges (Lam and Lee, 2012). In
these studies the added coagulants did not affect the cell membrane integrity or cause toxin release
from the cells during flocculation. However, extensive cell damage and release of intracellular
components 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 at an acidic pH during
water treatment, which photosynthetic organisms may not tolerate (Kim et al., 2011).
Polyionic polymers for coagulation
Formation of aggregates through the use of synthetic and organic polymers, i.e. polymer bridging,
has been investigated as an alternative to the use of metal salts, with some success. Synthetic
cationic polymers such as Praestol (1 mg/l) (Pushparaj et al 1993), PEI (20-30mg/l) (Zeleznik et al
2002, Arrington et al 2003) and polyacrylamide (3 mg/l) (Jancula et al 2011) are able to flocculate
cyanobacterial cells with between 80% and 90% efficiency of cell removal. Praestol did not affect
cell membrane integrity but PEI was shown to increase cell permeability.
The effect of
polyacrylamide on the cell viability was not tested. In addition to synthetic polymers, organic
flocculants such as clay and chitosan enhance the flocculation ability of cyanobacteria (Pan et al
2006a; Pan et al 2006b; Zou et al 2006; Verspagen et al 2006; Liu et al 2010; Divakaran n Pillai
2002). Whilst no adverse effect on cell membrane integrity has been demonstrated with chitosan
addition, the use of clay and chemically modified clay, especially chitosan modified kaolinite,
results in widespread death and lysis of cyanobacterial cells (Shao et al, 2012). Since the conditions
during floc formation such as temperature, ionic strength of the suspension medium, pH, strain type
and cell concentration differ between studies, the efficiency of the polymers in cell removal cannot
be directly compared. Moreover, the efficiency of cell-liquid separation using flocculation based
technologies is not consistent. It depends to a great extent, on the surface characteristics of the
suspended cells and the polymers present in the environment. These can be either natural organic
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matter (NOM) or polymers produced by the cells during growth – the extracellular polymeric
substances (EPS), also known as algogenic organic matter (AOM) (Henderson et al 2010; Teixeira
et al 2010).
Microbial extracellular polymeric substances (EPS) for coagulation
Microbial EPS are categorized in two separate fractions based on proximity to the cell surface, EPS
positioned near the cell surface by non-covalent interactions are termed “bound” EPS and those that
are secreted into the culture medium are called “free” or “released” EPS (Eboigbodin et al 2008).
The aggregation of cells within a biofilm is known to be aided by the favorable interactions between
physico-chemistry of the cell surface and the EPS (Karunakaran and Biggs, 2011). However, when
using coagulants, especially polyvalent metal salts, to induce aggregation, EPS of Aphanothece
halophytica and Microcystis aeruginosa increase coagulant demand (Henderson et al 2010; Chen et
al 2009; Chen et al 2010; Takaara et al 2007; Takaara et al 2010). On the other hand, the presence
of EPS, without the use of metal salts, can induce aggregation. The bioflocculation of kaolinite
using released EPS isolated from cultures of Phormidium sp., Anabaena circularis, Lynbyga sp.,
and Microcoleus sp. Was previously reported (Levy 1990; Levy 1992; Chen et al 2011). Recently, a
role of EPS in flocculation was proposed for Synechocystis sp., PCC 6803 (Jittawuttipoka et al
2013). Moreover, bioflocculant activity is not limited to released cyanobacterial EPS (Nie et al,
2011; Taniguchi et al 2005; Kim et al 2011). Interestingly, the bound EPS of cyanobacterial and
heterotrophic cells has also been indicated to aid flocculation. In species such as Arthrospira
plantensis, Tolypothrix tenuis and Desulfovibrio oxyclinae, autoflocculation is induced when the
cells are exposed to environmental stress (Markou et al 2012, Silva and Silva 2006, Sigalevich et al
2000). Acinetobacter calcoaceticus, a water isolate, will not only autoflocculate but also enhance
the flocculation ability of other bacteria (Simoes et al 2008). In addition, the bioflocculation of
algae using EPS does not impact on cell membrane integrity (Lee et al 2009; Kim et al 2011).
In conclusion, the harvesting of biomass without affecting the integrity of the cells is an
important area of research within industrial biotechnology. Bioflocculation of cells is a balance
between the physico-chemical properties of the cell surface and EPS, and could be a preferable
alternative to chemical coagulants. However, to facilitate biomass harvesting using bioflocculants at
the industrial scale a rigorous study of the cell surface characteristics and EPS production of the
cells under relevant operating conditions has to be carried out, especially because cell surface and
EPS have been shown to be affected by the environmental conditions (Eboigbodin et al 2006;
Eboigbodin et al 2007; Mukherjee et al 2012). Overall, there is an urgent need for
in-depth
characterization of surface properties and of EPS in photosynthetic organisms so that suitable cellwater separation technologies can be developed.
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DESIGN OF AN INTEGRATED PROCESS
The overall aims of municipal wastewater treatment plants (WWTPs) and water treatment plants
(WTPs) are to protect public health in a manner compatible with environmental, economic, social
and political concerns. Wastewater treatment commonly utilizes biological processes relying on
microorganisms to take up dissolved organic matter and nutrients. These processes take advantage
of the fact that microorganisms are relatively easy to remove through settling or filtration.
Biological treatment technologies deployed in wastewater treatment include the activated sludge
process (aerobic suspended-growth), trickling filters (and other attached-growth biological filters)
and membrane bioreactors (membrane filtration combined with a suspended growth bioreactor).
More advanced configurations of the activated sludge process, incorporating aerobic and anoxic
zones, can be operated for nutrient removal. There are increasing regulatory pressures, such as
contained in the EU urban wastewater treatment directive (EU, 1991), to limit nitrate and phosphate
contents with the aim to protect downstream aquatic ecosystems from eutrophication. This is
achieved through nitrification, denitrification and phosphate uptake by different communities of
bacteria. Other biological technologies used in WWTPs are aerobic lagoons and various suspendedand fixed-growth anaerobic processes (including a range of anaerobic digester and anaerobic filter
designs).
Reactors designed to promote viability, functionality and high concentrations of
photosynthetic organisms may differ significantly from those used in biological wastewater
treatment, even if both are based on principles of attached- and suspended-growth. Evidently, light
is a key parameter and reactors used to grow algae may prove more suitable in this respect. Many
photobioreactor designs are only used at laboratory scale and recent advances in light-emitting
diode (LED) technologies offer an opportunity to efficiently supply the requisite wavelengths of
light for photosynthesis. However, at full-scale this becomes less feasible in terms of operational
and capital cost, with a key challenge being to provide and regulate light exposure to photosynthetic
organisms. Large-scale open lagoons, are an appropriate system to achieve this. In common with
many engineered algal cultures, these are more favorable in locations with year-round high solar
radiation and temperature (Su et al., 2011). Nonetheless, many design improvements are still
needed in order to improve robustness, reduce energy consumption and optimize growth conditions
for large-scale production of photo-autotrophs. Providing a feed with the appropriate nutrient
profile, suitable temperature and mitigating against interference from other indigenous
microorganisms are other key challenges linked to a transition from growing photosynthetic
organisms at laboratory-scale to at industrial-scale. Of the nutrients required for photoautotrophic
growth, carbon dioxide is considered as the most significant, due to the high proportion (~50% of
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dry weight) of carbon in the biomass of photoautotrophic organisms (Kim et al., 2011). Large-scale
growth of photoautotrophic organisms relies upon huge amounts of carbon dioxide, which must be
delivered in an energetically-efficient manner. At laboratory scale this can be easily provided by
sparging with air and/or carbon dioxide. Bubble-less gas-transfer membranes, widely used in the
food industry, shows promise for larger-scale delivery of carbon dioxide (Kim et al., 2011). Overall,
in order to achieve improved reactor design it is critical to better understand the kinetics of nutrient
acquisition and photon capture by relevant organisms, so that their growth and rates of
photosynthesis can be properly controlled.
A crucial operational issue common to both wastewater treatment and growth of
photosynthetic organisms is delivering a sustainable and cost-effective disposal or reuse route for
the large volumes of biomass that will be inevitably produced. Promising avenues to achieve this
exist. Notably these include anaerobic digestion, biofuel production or utilising as a feed substrate
in aquaculture. With respect to biofuel production, genetically-modified strains of Synechocystis
PCC 6803 have been grown which secrete energy-rich fatty acids (Liu et al., 2011). Experience
from disposal of sewage sludge shows there are a number of challenges which will need to be
overcome before reuse of waste biomass from photosynthetic organisms will become viable. These
include effective low-energy dewatering and complying with the relevant legislation for reuse and
disposal of biosolids, likely to be a particular issue for genetically-modified biomass. The impact of
residual salt on downstream reuse applications also requires consideration. Whilst anaerobicallydigesting biomass has the major benefit of generating methane, a potential energy source, algal
sludge tends to be of relatively low biodegradability and methane yield (Bond et al., 2012). In such
situations pre-treatment or hydrolysis, to increase biodegradability, and/or co-digestion with a
complementary feed source are possible methods to improve digester performance.
The design of clarifiers for effective separation of photosynthetic organisms from water is
another important issue to consider when moving from laboratory scale to full scale of operation.
Sedimentation and flotation are two economically viable cell-liquid separation techniques typically
employed in WTPs. Both approaches require coagulation and efficient floc formation to achieve
high separation efficiencies. Sedimentation has the advantage of low capital expenditure and low
energy consumption during operation when compared to flotation. However, the ability of
cyanobacteria such as Synechocystis PCC 6803 and Synechococcus PCC 7002 to rise to the surface
of an open container (Figure 4) suggests that flotation strategies such as dissolved air flotation
(DAF) can help achieve high separation efficiencies rapidly.
ASSESSMENT OF RISKS AND PUBLIC ACCEPTANCE
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Any application of biodesalination technology has numerous health and environmental protection
issues that must be addressed during the design, construction and operation of the facility (WHO
2007). In addition, the use of synthetic biological applications, particularly involving cyanobacteria
with its known toxicity risks (Hunter et al. 2012), brings with it the risk of low social acceptance
(Bubela et al. 2012). Indeed, the general public has historically been skeptical about adopting
alternative water sources in general (Dolnicar et al. 2010) and proposed schemes have even been
abandoned due to a lack of public acceptance (Hurlimann and Dolnicar 2010; Hurlimann and
McKay 2007; Po et al. 2003). Much research has been undertaken into public acceptance of
recycled water, particularly in countries such as Australia where serious droughts, with their
accompanying severe water restrictions, have led to the search for alternative water supplies. More
recently, researchers have begun to also investigate public acceptance of desalinated water and have
discovered different degrees of acceptance, for both recycled and desalinated water, depending
upon the particular use intended (Hurlimann and Dolnicar 2011). Greater acceptance of desalinated
water, as opposed to recycled water, has been found for close-to-body uses while, for uses not close
to the body, e.g. irrigation or industrial cooling, recycled water is preferred (Dolnicar and Schäfer
2009; Dolnicar et al. 2011).
Factors such as education, age, knowledge, income and gender influence acceptance levels
of recycled water (Dolnicar and Schäfer 2009). In general, the more formal an education received,
the greater the person’s knowledge about recycled water and the higher the probability of their
acceptance (Sims and Baumann 1974). Related to this factor, Baumann (1983) found that the better
educated respondents had a greater faith in science and technology and therefore a higher
acceptance. Similarly, Marks (2006) argues that effective public consultation promotes greater trust
in those responsible for the assessment and management of risks and Po et al. (2003) ascribe the
success of a number of water reuse projects to a great emphasis on public involvement and
education. As far as desalination is concerned, it has been noted that the knowledge level
concerning the technology is relatively low (Dolnicar et al. 2011) and thus increasing the public’s
knowledge could increase acceptance levels. Dolnicar et al. (2010) looked specifically at how the
provision of information about alternative water supplies affected public perception. They
concluded that hesitance to embrace such water is primarily driven by water quality concerns but
providing people with basic information about recycled and desalinated water increased their
likelihood of using these alternative supplies.
In addition to the general skepticism over the use of desalinated water, the use of synthetic
biological applications in the field of biodesalination, particularly those involving GM
cyanobacteria with their inherent risks (Henley et al. 2013), increases the danger of low social
acceptance. Historically, public opinion to what may be viewed as the (re)design of nature and the
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merging of biology with engineering has been negative (Bubela et al. 2012). As with the
introduction of recycled and desalinated water, however, the provision of accurate information on
the benefits and risks of the technology in the early stages of any proposed project is believed to be
critical, particularly concerning the image portrayed by the media, which can have an adverse
influence on acceptance levels (Bubela et al. 2012). Notwithstanding this, Christoph et al. (2008)
concluded that educating consumers does not necessarily result in greater support for genetic
modification since increased knowledge does not automatically imply support.
Despite the importance of public opinion to the success of emerging technologies, there
remains a paucity of studies in the literature on public perceptions of synthetic biology. The
majority of research has been undertaken on social acceptance of genetically modified (GM) food
products (e.g. Costa-Font et al. 2008; Dannenberg 2009; Siegrist 2008). It is frequently argued that
consumer rejection of such foods is the result of their introduction without any perceived benefits to
consumers, together with the portrayed risks of genetically modified organisms (GMOs) to the
environment (Frewer et al. 2004). Other factors, such as ethical and moral considerations, and trust
in both the scientists conducting the research and the regulatory system, are also important
determinants of consumer acceptance or rejection of the technology (Frewer 2003; Siegrist 2008).
In a study by Magnusson and Hursti (2002), it was discovered that age and gender, together with
level of education, had an impact on likely acceptance of GM foods with males and younger
respondents generally more positive. Meanwhile, Prokop et al. (2013) discovered that disease risk
resulted in significantly more negative attitudes towards GM products. However, with current
stringent regulations governing the use of synthetic biological applications, such concerns should be
minimised, especially if the public are kept reliably informed from the early stages of development.
One of the key considerations in the application of the biodesalination technology concerns
potential locations. Issues of saline waters, and the requirement for desalination to augment
supplies, are well known in the Gulf States and South America where conventional desalination
plants already exist (Dawoud 2005). Social acceptance of emerging technologies has been shown to
vary between countries. In particular, experience of serious drought and water restrictions in
Australia has led to less resistance to recycled or desalinated water in recent years (Dolnicar and
Schäfer 2009,) suggesting that public opinions are affected by personal experiences. Historically,
developing countries were less opposed to the concept of genetic modification. However, Frewer
(2003) noted an increasing resistance to the introduction of GM foods in developing countries due
to the activity of national government organizations that opposes the implementation of genetic
technologies in agriculture. Meanwhile, studies in Germany (Christoph et al. 2008) and Sweden
(Magnusson and Hursti 2002) found strong negative tendencies to the acceptance of genetic
modification with the main concern being uncertainty about possible long-term effects to the
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environment and human health. Acceptability was greater towards applications involving non-food
products, however, since they are seen to be more beneficial, less risky and ethically correct, a point
also noted by Sorgo et al. (2012).
A biodesalination process based on genetically modified cyanobacteria will present multiple
challenges from the point of view of social and regulatory acceptance. It has clearly more chance of
success in countries where desalination is already an accepted practice; and where GMOs are not
seen as a threat by both government and population. The process will have to ensure that it fulfils
all safety requirements for GMO approval. It will also have to prove that there is no danger coming
from the use of cyanobacteria and to deal actively with potential negative perceptions due to toxin
generation. Consequently, it seems advisable to explore initially combined uses of the low salinity
water and biomass in productive systems designed for saline arid environments.
CONCLUSIONS AND OUTLOOK
This paper has examined, using the specific example of biodesalination, the challenges and
opportunities associated with applications of cyanobacteria in water treatment, many of which are
pertinent to other biotechnologies. The key part of the conceptualised biodesalination process is to
employ a low-salt biological reservoir within the cyanobacteria as an ion exchanger. Uptake of salt
into these reservoirs would then be mediated by genetic and/or environmental manipulation of the
cyanobacteria. As exemplified by the strains Synechocystis sp. PCC 6803 and Synechococcus sp.
PCC 7002, cyanobacteria have a number of attributes which make them attractive for such
applications, as they are fast-growing, tolerant of a range of salt concentrations and amenable to
genetic transformation. Furthermore, since the primary metabolism of cyanobacteria is based upon
photosynthesis, nutrient requirements are minimal and active salt export during growth is powered
by sun light. Solar radiation can also be used to energize subsequent salt accumulation through
expression of retinal ion pumps such as halorhodopsin. Protocols for genetic manipulation of
cyanobacteria through natural transformation and conjugation have been developed. As other
biotechnological processes, biodesalination requires efficient separation of cells from water.
Coagulation is a suitable method, since this can remove up to 99% of cyanobacteria and chitosan
flocculants have no adverse impact on viability of cyanobacteria. The design and operation of an
integrated biodesalination process is likely to build on knowledge of both algal bioreactors and
wastewater treatment processes.
Notwithstanding these opportunities, challenges need to be overcome at each stage of the
proposed biodesalination process. Further research is needed to elucidate the impact of
environmental factors, including pH, temperature and nutrients, on salt transport in cyanobacteria.
A major bottleneck for easy genetic manipulation is the limited availability of vector backbones that
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enable flexible rearrangement of essential elements, and of robust promoters that can operate as
biological switches in non-native settings. Furthermore, separation of planktonic cyanobacteria
from water is difficult due their low density and molecular size, and the presence of EPS can have
contradictory effects on aggregation. Consequently, to fully optimize separation, more work is
needed to characterize the surface properties of both cyanobacteria and EPS. Finally the use of
synthetic biological applications to produce recycled water brings the risk of low social acceptance,
although this varies geographically and may increase with further education.
ACKNOWLEDGEMENTS
The authors of this article joined forces to develop methods for biodesalination following a
Sandpit event (Water For All Challenge, 2010) organized by the Engineering and Physical Sciences
Research Council (EPSRC). The financial support from the EPSRC for this work is gratefully
acknowledged.
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Figure legends
Fig. 1 Proposed usage of cyanobacterial cultures for water treatment.
A. Hypothetical water treatment station. Situated in basins next to the water source, sun-powered
cell cultures remove unwanted elements from the water. The clean water is separated from the cells
for human usages. The produced biomass is available for other industries. The proposed biodesalination process is based on the following steps. B: Photo-autotrophic cells divide to generate
high density cultures. C: The combined cell volume is low in salt due to transport proteins in the
cell membrane that export sodium using photo-synthetically generated energy. D: Through
environmental and genetic manipulation salt export is inhibited and replaced with transport modules
that accumulate salt inside the cells. This process is again fuelled by light energy. E. Manipulation
of cell surface properties separates the salt-enriched cells from the desalinated water.
Fig. 2: Pre-screening of cyanobacterial cultures for strain selection.
The effects of different media and environmental conditions on the performance of cyanobacterial
cultures can be tested under controlled conditions in the laboratory.
Fig. 3 Na+ transport and its energization in different phases of the proposed desalination
process.
In the culture growth phase (left) the cells generate a low-salt reservoir inside the salty environment
through active export of Na+ by endogenous transport proteins (light grey circles) across the plasma
membrane (PM). These are either directly fuelled by ATP (Na+-ATPases) or, in the case of Na+/H+
antiporters, exploit the pH gradient established by H+-ATPases (dark grey circle). Na+ export from
the cytoplasm (cyto) therefore relies on ATP and proton motive force generated from light energy
captured by photosystems (green box) and chemiosmosis (ATP-synthase, grey 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 through Na+permeable channel proteins (grey box).
Fig. 4 Cell-water separation can take advantage of the ability of cyanobacteria to float.
Visual appearance of initially-mixed cultures of cyanobacteria strains PCC6803 (top) and PCC7002
(bottom) left under ambient laboratory light (8 ± 2 µM) for 24 h (n=3).
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Fig. 1 Proposed usage of cyanobacterial cultures for water treatment.
A. Hypothetical water treatment station. Situated in basins next to the water source, sun-powered
cell cultures remove unwanted elements from the water. The clean water is separated from the cells
for human usages. The produced biomass is available for other industries. The proposed biodesalination process is based on the following steps. B: Photo-autotrophic cells divide to generate
high density cultures. C: The combined cell volume is low in salt due to transport proteins in the
cell membrane that export sodium using photo-synthetically generated energy. D: Through
environmental and genetic manipulation salt export is inhibited and replaced with transport modules
that accumulate salt inside the cells. This process is again fuelled by light energy. E. Manipulation
of cell surface properties separates the salt-enriched cells from the desalinated water.
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Fig. 2: Pre-screening of cyanobacterial cultures for strain selection.
The effects of different media and environmental conditions on the performance of cyanobacterial
cultures can be tested under controlled conditions in the laboratory.
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Fig. 3 Na+ transport and its energization in different phases of the proposed desalination
process.
In the culture growth phase (left) the cells generate a low-salt reservoir inside the salty environment
through active export of Na+ by endogenous transport proteins (light grey circles) across the plasma
membrane (PM). These are either directly fuelled by ATP (Na+-ATPases) or, in the case of Na+/H+
antiporters, exploit the pH gradient established by H+-ATPases (dark grey circle). Na+ export from
the cytoplasm (cyto) therefore relies on ATP and proton motive force generated from light energy
captured by photosystems (green box) and chemiosmosis (ATP-synthase, grey 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 through Na+permeable channel proteins (grey box).
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Fig. 4 Cell-water separation can take advantage of the ability of cyanobacteria to float.
Visual appearance of initially-mixed cultures of cyanobacteria strains PCC6803 (top) and PCC7002
(bottom) left under ambient laboratory light (8 ± 2 µM) for 24 h (n=3).
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