JBA-07012; No of Pages 15
Biotechnology Advances xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Phosphorus from wastewater to crops: An alternative path
involving microalgae
Alexei Solovchenko a,b,c,⁎, Antonie M. Verschoor d,e, Nicolai D. Jablonowski c, Ladislav Nedbal c
a
Department of Bioengineering, Faculty of Biology, Moscow State University, Moscow, Russia
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia
Institute of Bio- and Geosciences, Plant Sciences (IBG-2), Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
d
Wetsus, European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911MA Leeuwarden, The Netherlands
e
Duplaco b.v., Hassinkweg 31, 7556BV Hengelo, The Netherlands
b
c
a r t i c l e
i n f o
Article history:
Received 18 August 2015
Received in revised form 8 January 2016
Accepted 11 January 2016
Available online xxxx
Keywords:
Bio-fertilizer
Luxury uptake
Microalgal biotechnology
Polyphosphate
Wastewater treatment
a b s t r a c t
Phosphorus (P) is a non-renewable resource, a major plant nutrient that is essential for modern agriculture.
Currently, global food and feed production depends on P extracted from finite phosphate rock reserves mainly
confined to a small number of countries. P limitation and its potential socio-economic impact may well exceed
the potential effects of fossil fuel scarcity.
The efficiency of P usage today barely reaches 20%, with the remaining 80% ending up in wastewater or in surface
waters as runoff from fields. When recovered from wastewater, either chemically or biologically, P is often
present in a form that does not meet specifications for agricultural use. As an alternative, the potential of
microalgae to accumulate large quantities of P can be a way to direct this resource back to crop plants. Algae
can acquire and store P through luxury uptake, and the P enriched algal biomass can be used as bio-fertilizer.
Technology of large-scale algae cultivation has made tremendous progress in the last decades, stimulated by
perspectives of obtaining third generation biofuels without requiring arable land or fresh water. These new
cultivation technologies can be used for solar-driven recycling of P and other nutrients from wastewater into
algae-based bio-fertilizers.
In this paper, we review the specifics of P uptake from nutrient-rich waste streams, paying special attention to
luxury uptake by microalgal cells and the potential application of P-enriched algal biomass to fertilize crop soils.
© 2016 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Phosphorus in the environment and in the algal cell . . . . . . . . . . . . . . . . . . .
2.1.
Reservoirs and fluxes of phosphorus in geo- and agrosphere . . . . . . . . . . . .
2.2.
Extracellular phosphorus pools . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Measures for phosphorus reduction in surface waters . . . . . . . . . . . . . . .
2.4.
Opportunities for and advantages of using algae for phosphorus capture and recovery
2.5.
Inorganic phosphate uptake by algal cells . . . . . . . . . . . . . . . . . . . .
2.6.
Phosphorus pools in algal cells . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.1.
Integral phosphorus capacity of algal cells . . . . . . . . . . . . . . . .
2.6.2.
Storage polyphosphates . . . . . . . . . . . . . . . . . . . . . . . .
2.6.3.
Subcellular distribution of polyphosphates . . . . . . . . . . . . . . .
2.6.4.
Physiological roles of polyphosphates . . . . . . . . . . . . . . . . . .
2.7.
Strategies of Pi sequestration in algal cultivation systems . . . . . . . . . . . . .
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Abbreviations: AISP, acid insoluble polyphosphate; ASP, acid soluble pholyphosphate; DW, dry weight; EBPR, enhanced biological phosphate removal; PBR, photobioreactor; P,
phosphorus; Pi, inorganic phosphate; WSP, wastewater stabilization pond.
⁎ Corresponding author at: Faculty of Biology, Moscow State University, Leninskie Gori 1/12, 119234, GSP-1 Moscow, Russia.
E-mail address: [email protected] (A. Solovchenko).
http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
0734-9750/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
2
A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
3.
Phosphorus uptake under different cultivation conditions . . . . . . . . . . .
3.1.
Orthophosphate concentration . . . . . . . . . . . . . . . . . . . .
3.2.
Photosynthesis, irradiance, and temperature . . . . . . . . . . . . . .
4.
The potential of microalgal biotechnology for phosphorus recycling . . . . . . .
4.1.
Real-world phosphorus sequestration . . . . . . . . . . . . . . . . .
4.2.
Monoculture or association? . . . . . . . . . . . . . . . . . . . . .
4.3.
Providing algal strains for luxury P uptake . . . . . . . . . . . . . . .
4.4.
Choosing the cultivation mode for microalgal phosphorus sequestration .
4.4.1.
Open ponds . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.2.
Photobioreactors for suspension cultures . . . . . . . . . . .
4.4.3.
Immobilized microalgae . . . . . . . . . . . . . . . . . . .
4.4.4.
Thin-layer cultivation systems . . . . . . . . . . . . . . . .
4.5.
Production and use of the phosphorus rich microalgal biomass . . . . . .
4.5.1.
Production of phosphorus rich algal biomass using waste streams
4.5.2.
High-value P-rich products . . . . . . . . . . . . . . . . . .
4.5.3.
Algal bio-fertilizer . . . . . . . . . . . . . . . . . . . . . .
5.
Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Phosphorus (P) is a finite, non-renewable resource and a foundation
of modern agriculture (Elser, 2012). After nitrogen, P is the second most
frequent macronutrient that limits plant growth. It is an essential component of key molecules such as nucleic acids, phospholipids and ATP,
and consequently, plants cannot grow without a reliable supply of this
nutrient.
Currently, agriculture depends almost exclusively on P extracted
from phosphate rock (Cordell et al., 2009, 2011), which is not distributed uniformly over the globe. Morocco, China, Algeria, United States,
South Africa, and Jordan possess the majority of the world's minable P
rock, which is expected to be largely exhausted within this century
(Jasinski, 2012; Bennett and Elser, 2011; Cordell et al., 2009;
Obersteiner et al., 2013; Cordell and White, 2014). In a few of the production countries, the P rock reserves may last longer (Fixen and
Johnston, 2012) and changes in demand and other dynamic factors
can influence the supply (Scholz and Wellmer, 2013). However, from
a global perspective, the need to close the anthropogenic P-loop is
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urgent. The limited availability of phosphate rock contrasts with eutrophication (nutrient pollution) of natural waters by P washouts from
fields and by pollution from animal manure and other waste streams
(Fig. 1; see also Anderson et al. (2002)).
Global P and food security as well as the reduction of excessive nutrients of natural waters can only be achieved by synergistic measures that
combine increased P use efficiency, changing diets, and the re-use of
manure, human excreta, crop residues, and other waste (Cordell and
White, 2014). Phosphorus recovery from wastewater and its reuse is
particularly attractive (Carpenter and Bennett, 2011; Cordell et al.,
2009, 2011; Shilton et al., 2012). The chemical precipitation of P with
Al, Fe, or Ca compounds is already widely applied (Mulbry et al.,
2005). However, P from chemically precipitated sludge is not readily
bioavailable and is typically lost in landfills (de-Bashan and Bashan,
2004) although biological systems can mobilize chemically bound P to
a certain extent (Desmidt et al., 2015; Wilfert et al., 2015). Among alternative strategies currently in use, “enhanced biological phosphorus removal” (EBPR) stands out; in this process, P is removed from
wastewater through accumulation in heterotrophic sludge bacteria
Fig. 1. An image acquired by the Envisat satellite (Full Resolution, MERIS_FR) showing Lake Erie, USA in 2011. Phosphorus from farms, sewage, and industry facilitated a harmful algae
bloom that threatened public health due to hepatotoxin microcystin. The figure demonstrates the negative environmental impact of algae grown on P in runoff waters as well the
positive potential of algae to take up large quantities of P if handled properly. Adopted from https://earth.esa.int/web/earth-watching/environmental-hazards/content/-/article/algalblooms-in-lake-erie-north-america- with permission from the European Space Agency.
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
(Kulaev et al., 2004; McGrath and Quinn, 2003). EBPR is one of the most
investigated biotechnological processes due to its widespread application (García Martín et al., 2006). It is technically more complicated
than chemical P recovery, but the sludge is better suited for agricultural
application. Often EBPR is combined with chemical P recovery, for example by struvite (magnesium ammonium phosphate) precipitation
after anaerobic digestion of EBPR sludge (Desmidt et al., 2015; Wilfert
et al., 2015).
Wastewater treatment using photosynthetic organisms also has a
long history (Oswald, 1988, 2003; Oswald and Gotaas, 1957). The recent
impetus to this application is the dynamic development of microalgal
technologies focused towards industrial-scale production of biofuels
that could be made economically viable and sustainable when using nutrients from wastewater (Pittman et al., 2011; Wijffels et al., 2010). Due
to a number of significant advantages, microalgae-based techniques
also gain importance among the possible approaches to manage P in
wastewater. Microalgae can take up inorganic phosphate (Pi) at a rate
that allows them to successfully compete with bacterioplankton in natural conditions (Currie and Kalff, 1984) and accumulate P up to 2–3% of
their cell dry weight (Powell et al., 2009; Wood and Clark, 1988) — a
trait that can be applied to remove Pi from treated wastewater (Boelee
et al., 2011). The P-enriched microalgal biomass could then be converted, for example, to feed additives and bio-fertilizers, aiding P retention
and recycling (Mulbry et al., 2005). There are many added benefits,
such as the removal of nitrate, destruction of organic pollutants, suppression of pathogenic microflora, and CO2 sequestration1 (De Pauw
and Van Vaerenbergh, 1984; Pittman et al., 2011).
Phosphorus uptake and turnover in microalgae has been already the
subject of intensive research at diverse levels — from cells to ecosystems
(Cembella et al., 1984, 1982; Kulaev et al., 2004) and biosphere
(Carpenter and Bennett, 2011). Past research focused mostly on environments with low P availability and the mechanisms developed by microorganisms to cope with P limitation. In contrast, reviews dedicated to
the biology of microalgae in a P-rich environment are scarce (Brown
and Shilton, 2014; Powell et al., 2009). Polyphosphate is the main
form of P stored in algal cells, and in spite of its central role in many biological processes, we know little about it (Rasala and Mayfield, 2015).
In this paper, we review the specifics of P uptake from P-rich environments, such as waste streams, as well as strategies for the sequestration and recycling of this element. We pay special attention to luxury
uptake of P by microalgal cells, and the potential to convert algae into
bio-fertilizers with slow release of P.
2. Phosphorus in the environment and in the algal cell
2.1. Reservoirs and fluxes of phosphorus in geo- and agrosphere
In terms of mass, the P concentration is approximately 1180 ppb in
the lithosphere and 70 ppb in the seawater (Fairbridge, 1972), ranking
it 10th and 19th, respectively, of the most abundant elements on
Earth. The P concentration in the biosphere is estimated to be approximately 250 ppm (Cole and Rapp, 1981), an amount that is also relatively
low when one considers the essential biochemical role of the element:
no genetic biological information transmission, no biological energy
transduction, and thus no life in its present form would be possible
without P (Rasala and Mayfield, 2015).2
Although the total pool of P in the Earth's crust is large and its concentration in the biosphere relatively small, biomass and productivity
of primary producers are often P limited (Elser, 2012; Elser et al.,
3
Fig. 2. A schematic representation of different P pools and fluxes inside (left) and outside
(right) of an algal cell. Outside the cells, Pi is either dissolved in water or bound to the cell
wall. In this chemical form, P is directly available for active transport into the cell. The
other two forms, dissolved organic P (DOP) and colloidal P, can be converted to Pi by
extracellular enzymes (Grobbelaar, 1983). The box in the lower right corner represents
the fraction of biologically unavailable P, that is not available to enzymatic conversion.
Inside the cell, Pi is used for the synthesis of phospholipids, which are primarily
constituent components of cell membranes, to P sugars, which are also building blocks
of nucleotides and nucleic acids (DNA and RNA). (De-)phosphorylation is also an
important process regulating phosphoproteins. Phosphorus is stored in the form of 4
different types of polyphosphates (polyphosphate A–D), which are constituted
depending on light and Pi availability. These reserves can be mobilized differentially
into nucleic acids, P lipids, or into Pi. Formation, interconversion, and catabolism of
different polyphosphate pools in Chlorella under P-replete (open arrows) and Pstarvation (closed arrows) A, C: acid-soluble (A: extractable by 8% trichloracetic acid; C:
precipitated by neutralization of a 2N KOH extract with HClO4 in the presence of KClO4;
B, D: alkali-soluble (Miyachi and Miyachi, 1961; Miyachi and Tamiya, 1961a; Miyachi
and Tamiya, 1961b).
2007; Smil, 2000). This discrepancy is partially due to: 1) the low content of P in cellulose, hemicellulose, and lignin, which represent a
large share of biomass; and 2) the low bioavailability of P in poorly soluble rock apatite, which accounts for 95% of P in the Earth's crust. The
natural P cycle depends on the tectonic uplifting of rocks and exposure
to weathering, typically on a timescale of 107–108 years. Fast atmospheric transfers of the element similar to carbon and nitrogen are absent because there are no long-living gaseous P compounds that could
facilitate a faster and more uniform P distribution over the globe. Cycling of soluble P compounds is limited by rapid precipitation in aluminum (low pH) or calcium (high pH) containing complexes.
The slow natural P cycle abruptly increased because of the massive P
mining for agricultural fertilizer production that facilitated the Green
Revolution of the twentieth century and continued to increase thereafter (Cordell et al., 2011). The present massive anthropogenic transfer of
P from rock to agriculture is, unfortunately, not a sustainable solution
because the minable mineral reservoir will be exhausted in the next
several decades as the human population and the demand for quality
nutrition continue to increase at an unprecedented pace (Elser et al.,
2007; Lima et al., 2003).
2.2. Extracellular phosphorus pools
1
Q7
By CO2 sequestration, we mean the process in which gaseous CO2 from the atmosphere or other sources is assimilated into algal biomass. One needs to remember, however, that the carbon fixed in the algal fertilizer can be respired back to atmospheric CO2 after
application to the soil.
2
A failed effort to identify a substitute for phosphate in arsenate is discussed in Reaves
et al., 2012.
P compounds are often classified based on their reactivity with molybdate, ease of hydrolysis, and particle size. The fractions capable of reaction with acid-molybdate to form a heteropoly blue complex are
generally termed “reactive phosphate”. Unfortunately, this classification
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
4
A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
is derived from an arbitrary physical–chemical characterization that is
poorly related to the biological significance of the P species (Cembella
et al., 1982). With respect to availability to living organisms, P species
in the environment are divided into two groups: bioavailable or nonbioavailable (Fig. 2). Algae can take up Pi directly from their
environment3 (Atlas et al., 1976). More complex bioavailable P compounds are decomposed to Pi by extracellular or cell-wall-bound phosphatase enzymes (see Fig. 2). In such a process, for example, dissolved
organic P (DOP) is hydrolyzed by alkaline phosphatase, phosphodiesterase, and phytase (Herbes et al., 1975; see also Table 1 in Cembella
et al., 1982). As an interesting additional pathway, some cyanobacteria
such as Trichodesmium can prevail in low P, oligotrophic waters by taking up P directly in form of naturally occurring phosphonate (Dyhrman
et al., 2006).
Concentrations of individual P species in natural water are highly
variable (Paytan and McLaughlin, 2007). The concentration of soluble
reactive P in deep, aphotic zones is approximately 1–3 μM. Towards
the ocean surface, it is depleted by phytoplankton uptake. Therefore, P
often becomes a limiting macronutrient in oligotrophic waters
(Thingstad et al., 2005). The concentration of P (and N) in natural waters can be increased by washout from fields and wastewater efflux
(Carpenter et al., 1998; Correll, 1998), causing noxious algal and
cyanobacterial blooms (Conley et al., 2009; Schindler, 1977).
Typical municipal wastewater concentrations of P rose in developed
countries from 2 to 4 mg L−1 in the 1940s to 10–15 mg L−1 20 years
later (Litke, 1999; Rybicki, 1997). This led to the massive increase in organic and nutrient loading in ground and surface waters that occurred
during the 1950s and 1960s. In the same period, algae blooms accompanied by shifts in phytoplankton community composition occurred in
lakes and rivers (Fig. 1). Some of the blooms contained toxic algal species. These phenomena entailed changes in both water turbidity and
the abundance and composition of macrophytes, invertebrates, and
fish. For surface waters worldwide, correlations were found between
chlorophyll concentration, microalgae abundance, and nutrient loading,
particularly P (Vollenweider, 1968). Although the debate continues
about which nutrient is predominantly responsible for this eutrophication, it is clear that P plays an important if not the central role in anthropogenic stimulation of algal growth (Schindler, 1977; Schindler et al.,
2008).
2.3. Measures for phosphorus reduction in surface waters
In response to the pivotal role of P in the eutrophication process,
three abatement policies developed in order to improve water quality.
In Europe, the earliest water legislation (1975) included standards for
surface water intended for drinking and quality rules for other water,
such as bathing water or groundwater. In 1991, the European Union
adopted two important directives to regulate the quality of ground
and surface waters of member states: the Nitrate Directive (nitrate pollution from agriculture) and the Urban Waste Water Treatment Directive. Under the latter directive, effluents discharged into sensitive
freshwater areas needed to be treated so that either an 80% reduction
in Pi was achieved or that the final Pi concentrations would be between
1 and 2 mg L−1. Later, new legal measures, such as the Bathing Water
Directive (2006) and the Drinking Water Directive (1998), were
adopted for sensitive water types. To better integrate and coordinate
policies related to water quality management, the EU adopted the
Water Framework Directive using river basins as the basis for water
quality management (Directive 2000/60/EC of the European Parliament
and of the Council establishing a framework for community action in
the field of water policy). This framework set guidelines and measures
depending on the water type to achieve a desired quality level. For
3
Inorganic phosphate Pi includes anions of orthophosphoric acid that can be phosphate
2–
–
PO3–
4 , hydrogen phosphate (HPO4 ) or dihydrogen phosphate (H2PO4) under strongly basic conditions, weakly basic conditions or weakly acidic conditions, respectively.
example, the Dutch great rivers are designated as highly changeable
waters, for which a summer average of 0.14 mg L− 1 P would be required. For sensitive waters, stricter regulations apply. Similarly, the
United States Environmental Protection Agency, the Canadian Council
of Ministers of the Environment (CCME, 2004, 2007), and the
Australian and New Zealand Guidelines for Fresh and Marine Water
Quality (ANZECC, 2000) have implemented type-specific water quality
regulations.
Two source-oriented measures proved to be highly effective in the
1980s, when approximately 80% of the detergent P load originated
from domestic wastewater (Rybicki, 1997). The first was the ban on
the use of P in detergents and soaps, causing the per-capita detergent
P load to surface water to drop from 0.5–1.1 kg y− 1 P in the 1970s
down to 0.2 in the 1990's (De Jong and de Oude, 1988; Rybicki, 1997).
The second was increasing the efficiency of P removal in domestic
wastewater treatment plants. The contributions of human excreta to
domestic effluent have been estimated to be 0.5 kg y−1 P per capita,
of which two thirds can be attributed to urine and one third to feces
(Rybicki, 1997). In a typical treatment plant, only 40–50% of the P can
be removed from the liquid phase, whereas more advanced P removal
techniques targeting the sludge can lead to a total removal of over 90%
of the influent load at a wastewater treatment plant (Desmidt et al.,
2015). Phosphorus removal from wastewater is achieved by chemical
and biological processes. Chemical phosphorus removal (CPR) is based
on the addition of trivalent cations, typically iron (Fe) or aluminum
(Al) salts, which form insoluble precipitates with phosphorus
(Desmidt et al., 2015; Wilfert et al., 2015). Biological P removal is
most often done by EBPR (Mino et al., 1998). To recover the P from
digested EBPR sludge, magnesium salts are used for struvite formation
(Desmidt et al., 2015; Wilfert et al., 2015). The efforts to reduce P in surface waters were particularly noticeable in rivers such as the Rhine,
where a sharp decrease in P levels was observed in the 1980s after the
introduction of EBPR and the ban on P in detergents and elimination
of several of its major sources (Fig. 3).
2.4. Opportunities for and advantages of using algae for phosphorus capture
and recovery
The use of algae for P (and nitrogen) removal and recovery has been
promoted over the past decades (de-Bashan and Bashan, 2010; Doran
and Boyle, 1979; Nesbitt, 1966). Recently, the efforts towards this goal
have intensified (e.g. de-Bashan and Bashan, 2004 or Hernández et al.,
2013; Sawayama et al., 1998; Shilton et al., 2012; Solovchenko et al.,
2013, 2014), stimulated by:
1. the increasing need for lowering the nutrient levels further, particularly for sensitive water types;
2. the economic and ecological arguments against using chemicals,
such as iron or aluminum, at wastewater treatment plants;
Fig. 3. Annual flow-weighted mean concentrations of NO3–N and PO4–P in the Rhine at the
German–Dutch border (Lobith). Reproduced from Stålnacke (2004) with permission.
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
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A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
5
3. the need to decrease the environmental footprint of wastewater
treatment facilities (Shilton et al., 2012);
4. the capacity of algae to operate in an aerobic environment, unlike
bacteria in EBPR.
Fig. 4 schematically shows that several relevant P-rich waste streams
may be accessible for sequestration by algae (dotted arrows). The
scheme is consistent with the estimates of Cordell et al. (2009), who
suggested the P loss from animal manure amounts to approximately
40% of the annual P mining quota, from human excreta it is up to 10%,
and from other industrial uses and products such as detergents, it accounts for almost 10%. These figures, considered in the context of P
rock limitation (Cordell and White, 2014), makes recovering P from
waste flows an imperative.
2.5. Inorganic phosphate uptake by algal cells
The negative charge of the Pi ions prevents its spontaneous diffusion
across the lipid bilayer of the algal cell membrane, which is not only hydrophobic but also negatively charged on its internal side (Fig. 2). Similar to fungi (Burns and Beever, 1979) or bacteria (Rosenberg et al.,
1977), kinetic and biochemical studies of Pi uptake in microalgae
suggest at least two distinct mechanisms of Pi transport across the
plasmalemma: one is activated when the Pi concentration in the environment is low and the other when Pi is abundant. Detailed molecular mechanisms of these uptake processes in microalgae remains
scarcely known.
According to the ample evidence reviewed in Cembella et al. (1982,
1984), Pi uptake is an active process occurring at the expense of ATP hydrolysis and/or membrane potential energy and involving co-transport
of cations (most probably H+ or Na+). In low-P environments, this
energy-intensive process can be facilitated by increasing the bioavailable P concentration in the environment through extracellular alkaline
phosphatase (see Fig. 2) or by enzymes excreted by other microorganisms. In P-rich environments, passive diffusion can also take place. For
a comprehensive account of the general biology of P uptake and metabolism in microalgae, we refer readers to reviews by Cembella et al.
(1982, 1984) and Grossman and Aksoy (2015).
To estimate the suitability of algae for EBPR, one must compare the
efficiency of P uptake systems in algae with that of other biotechnologically established microorganisms capable of both inorganic (Pi) and organic (DOP) P uptake (Blank, 2012). The primary Pi uptake occurs in the
Escherichia coli model by the phosphate-specific transporter (PST) family (Hsieh and Wanner, 2010) that consumes 2 ATP per one Pi molecule.
In a nutrient-rich environment, P is assumed to be transported by lowaffinity metal transporters driven by a transmembrane proton gradient
and a transport-neutral phosphate complex with divalent ions such as
Mg2+, Mn2+, Ca2+, and Co2+.
Unlike in E. coli (Hsieh and Wanner, 2010), fungi, and higher plants
(Bucher, 2007; Rausch and Bucher, 2002; Tian et al., 2012), the molecular nature of Pi in microalgae transporters remains fragmentary, relying
mainly on genomic data mining. For example, a sodium-dependent Pi
transporter gene, DvSPT1, was isolated from a cDNA library of Dunaliella
viridis (Li et al., 2006) similar to that of TcPHO from Tetraselmis chui.
More detailed information on Pi transporters is available for a model
microalga Chlamydomonas reinhardtii (Grossman and Aksoy, 2015).
Similar mechanisms of (active) Pi transport are assumed to operate in
the membranes of cell organelles, for example, in chloroplasts that
could resemble those of higher plants (Pfeil et al., 2014).
The P uptake by algae can respond dynamically to changes in nutrient availability. Currently there are two known dynamic modes of
P uptake in microalgae in P-rich environments. The first mode, overcompensation or “overshoot” (Cembella et al., 1982), occurs in prestarved algal cells after exposure to a P-rich environment. The transfer results in a rapid, light-dependent accumulation of acid-soluble
polyphosphate (Aitchison and Butt, 1973). The second uptake mode,
Fig. 4. A schematic representation of potential algae-based interventions in key P-flows in
the anthroposphere. The flows are represented relative to the annual quota of P-rock
mining (100%, 18 Mt(P) y−1 (Cordell et al., 2009)). On the source side of the
anthroposphere, the annual P-fertilizer application represents 78% and grazing 67% of
the mining quota. The black arrows indicate losses of P occurring in all steps from
mining (yellow), processing in the food and feed chains (green), industrial use (gray),
and waste chains (brown). The losses occur in forms that may not be accessible to
sequestration by algae because of involved chemical or physical forms or because of low
concentration levels. In contrast, the P-rich waste streams (red) that can be considered
for phosphate sequestration by algae are massive, capped as high as 64% of the annual
mining quota. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
luxury uptake, was originally identified in studies relating growth to
nutrient availability. One of the early seminal works on P storage in
algae (Ketchum, 1939) showed that the N:P uptake ratio can change
and also that the N:P ratio in biomass varies with the concentration of
both nutrients in water. Ketchum concluded that “phosphorus must
also be utilized in reactions which do not involve nitrogen,” suggesting
P storage mechanisms. Recently, a mathematical model approached the
N/P co-limitation (Bougaran et al., 2010), and Saito et al. (2008)
discussed the underlying concepts. For example, Pi availability in the
low concentration range affects not only P uptake and storage but also
cell quota of nitrogen.
Importantly, no pre-starvation is required for luxury uptake (Eixler
et al., 2006), which is a storage of P reserves occurring even when
there is a sufficient supply of it in the environment. Luxury P uptake
probably evolved as an adaptation of microalgae to unstable P availability. Watanabe et al.'s (1988) observations that the microalga
Heterosigma akashiwo adjusts its Pi metabolism during its vertical migrations support this hypothesis. At night, this alga migrates to the
lower, Pi-rich water layer and absorbs the nutrient, which is incorporated in polyphosphate, increasing its chain length. During the day, when
the alga migrates to the Pi-depleted surface water, it takes Pi from its
polyphosphate storage for photophosphorylation. De Mazancourt and
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A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
Schwartz (2012) also suggested that luxury uptake was a competitive
strategy aiming at starving the competitor.
2.6. Phosphorus pools in algal cells
2.6.1. Integral phosphorus capacity of algal cells
The Pi concentrations inside cells are typically close to 5–10 mM
(Brown and Kornberg, 2004; Rasala and Mayfield, 2015), independent
of external concentrations that may vary from the low micromolar
range in the deep aphotic ocean up to the low millimolar range in
wastewater effluent (see above). The Ki for Pi uptake is below 4 μM for
most species; this fact is believed to indicate the fundamental similarity
of the mechanism(s), presumably active transport, involved in Pi uptake
in different algal classes under a wide range of environmental conditions (Cembella et al., 1982).4 Although the intracellular Pi concentrations are modest, its turnover is immense (Blank, 2012).
The stoichiometric ratio of the essential elements carbon, nitrogen,
phosphorus, and sulfur is largely conserved in marine phytoplankton,
close to the Redfield ratio (C : N : S : P) of 106 : 16 : 1.7 : 1 (gravimetric
ratio 41:7.2:1.75:1) (Redfield, 1958). In comparison, the relative content of P in land plants is much lower because of the large C-content
in the phytomass (Smil, 2000). Using the Redfield ratio, one can estimate a typical gravimetric share of P in dry weight of natural phytoplankton at approximately 0.9%, corresponding to b1% estimate of
(Grobbelaar, 2004, 2013). However, the dry weight (DW) relative content of P in microalgae fed by swine manure can reach up to 1.8%
(Kebede-Westhead et al., 2006; Pizarro et al., 2006), and levels up to
4% DW of P have been reported in Powell et al. (2011a). This confirms
that microalgal cells can take up much more P than necessary for the
next life cycle (Rhee, 1973; Tilman and Kilham, 1976). A rough calculation of the potential of algae non-optimized P uptake capacity leads to
an estimate that a production area of approximately 26,600 km2 (a little
less than the area of Albania) would be required to sequester the 7 Mt of
P that is annually lost worldwide from animal manure (Smil, 2000). This
calculation assumes a content of 1.8% P in dry weight (KebedeWesthead et al., 2006; Pizarro et al., 2006) and algal productivity in an
open, thin-layer system (40 g (DW) · m− 2 · day− 1 (Doucha and
Lívanský, 2014). The required area would be proportionally even smaller with a higher P cell content (Powell et al. (2011a)).
In order to assess algae sequestering potential, one must investigate
the fluxes of the element in a cell surrounded by a Pi-rich medium.
When Pi first enters the cell, a considerable part of it is consumed for
the biosynthesis of cellular functional building blocks (function in
Fig. 2) or stored for a possible P shortage (storage in Fig. 2). Among
the functional structures, nucleic acids serve primarily for containing
and transferring biological information, and P is an indispensable part
of their polymer structure. Ribosomal RNAs, which are the most abundant nucleic acid molecules in the cell (Blank, 2012) and play an essential role in protein biosynthesis, make a prominent contribution to the
cell P quota (Tian et al., 2012). The latter feature may be of particular importance for the rapid Pi uptake from P-rich environments by fastgrowing algal species; they likely require a higher number of ribosomes
per cell compared to slow growers.
A functionally important pool of Pi is involved in phosphorylation
and de-phosphorylation of proteins that serve a crucial regulatory role
(Wang et al., 2014; Rochaixm, 2014; Ruelland et al., 2015). Phospholipids are essential structural elements of biological membranes
(Khozin-Goldberg and Cohen, 2011). Phosphatidyl inositol is present
in the plasma membrane of microalgae, being involved in signaling
(Testerink and Munnik, 2005).
4
Interestingly, the species with higher P uptake rates (in P-starved cultures) are encountered more often among dinoflagellates than among chlorophytes; see Cembella
Q15 et al., 1982.
Phosphorus also plays an indispensable role in energy storage and
transduction. The short-term energy storage and effective “energy currency” are the triphosphate bonds of ATP, which is a ubiquitous energy
storage form. ATP plays a central role in algae because it is the primary
product of photosynthesis. A considerable part of intracellular P is also
associated with different P metabolites, primarily sugar phosphates
formed with direct or indirect participation of ATP and other energyrich phosphorylated compounds.
The typical mid- and long-term energy storage compounds are carbohydrates or lipids, but significant free energy is also often contained
in polyphosphate, which contains tens to hundreds of bonds similar to
ATP. Because of its crucial role in P metabolism of algal cells, we discuss
polyphosphate in more detail below.
2.6.2. Storage polyphosphates
Through luxury uptake, microalgal cells can take up much more P
than is necessary for their immediate growth. The intracellular (cytosolic) Pi may be stored in polyphosphate pools deposited in vesicles or vacuoles (storage in Fig. 2). Polyphosphates serve as an
internal P depot that cells can use when needed (Kuhl, 1974;
Nishikawa et al., 2006). Enzymologists and biochemists have studied
polyphosphate synthesis in prokaryotic and eukaryotic heterotrophic organisms (for example, in yeast) relatively well (Wood and
Clark, 1988). By contrast, the data on the mechanisms of the biosynthesis and degradation of polyphosphates in phototrophic eukaryotic cells are scarce even though they are the most abundant P reserves
in microalgal cells (Cembella et al., 1982; Martin et al., 2014; Powell
et al., 2009).
For a better understanding of the role of polyphosphates in
algae, one must consider their broader role in nature and biology.
Achbergerová and Nahálka (2011) proposed that polyphosphate
formed on the prebiotic Earth through dehydration of phosphate rock
at high temperature. Polyphosphates were tentatively proposed as the
main energy storage molecule in early evolution (Achbergerová and
Nahálka, 2011), and if this is true, it is not surprising that polyphosphate
became widespread or even ubiquitous in pro- and eukaryote cells
(Brown and Kornberg, 2004; Kulaev et al., 2004; Nishikawa et al.,
2006; Rasala and Mayfield, 2015). The exceptions to the widespread occurrence of polyphosphates are also interesting. The heterokontophyte
Chattonella antiqua as well as Pylaiella sp. are reportedly unable to synthesize polyphosphates (Kimura et al., 1999) although one ought to
keep in mind the comment by Raven and Knoll (2010) that “inability
to detect polyphosphate in a given cell may be a problem of technique.”
Miyachi and colleagues (Miyachi et al., 1964; Miyachi and Miyachi,
1961; Miyachi and Tamiya, 1961a, 1961b) differentiated at least four
fractions of polyphosphate in algal cell. The different polyphosphate
pools are linked as P donors to specific metabolic pools depending on illumination conditions and cell cycle phase. During the life cycle of
microalgae in synchronous cultures, the newly absorbed Pi was first discovered in polyphosphate C, then in polyphosphate A, nucleotidic phosphate, and in DNA (Miyachi and Tamiya, 1961a). In Fig. 2, A and C
represent the more readily available acid-soluble polyphosphate (ASP)
fractions that are generated when light and Pi are abundant and that
can transfer Pi to DNA and protein. Fraction A was found in volutin
granules, named after the bacterium Spirillum volutans, in which polyphosphate was discovered as metachromatic granules in the cytoplasm
(reviewed by Achbergerová and Nahálka, 2011). Fraction A's accumulation depended largely on photosynthesis, probably because it is derived
from fraction C, which is presumably localized either in chloroplasts or
their vicinity. Fraction C donates Pi for the biosynthesis of chloroplast
DNA, whereas fraction A is involved in the synthesis of nuclear DNA
(Miyachi and Miyachi, 1961; Miyachi and Tamiya, 1961a).
The biosynthesis and degradation of alkali-soluble and acidinsoluble polyphosphate (AISP) fractions B and D rely on alternative energy reserves rather than on photosynthesis, and the cell cannot use
these fractions as readily (Fig. 2). The AISP fractions B and D serve as
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
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endogenous Pi reservoirs that are used in the absence of external Pi. The
prominent role of polyphosphate B is in the synthesis of RNA in the dark
and in low Pi conditions (Fig. 2).
2.6.3. Subcellular distribution of polyphosphates
Polyphosphates are in different compartments of microalgal cells. The
presence of polyphosphate in the volutin granules of Auxenochlorella
pyrenoidosa (earlier Chlorella pyrenoidosa) and Scenedesmus fuscus (earlier Chlorella fusca) was demonstrated using electron microscopy (Adamec
et al., 1979; Atkinson et al., 1974). With a more advanced electron microscopy technique known as X-ray microanalysis, researchers were able to
locate granules containing P in the cytoplasm, vacuoles, and chloroplasts
of Scenedesmus quadricauda (Voříšek and Zachleder, 1984). The cells of
microalga, such as C. reinhardtii (Ruiz et al., 2001), possess special
polyphosphate and Ca2+ storage organelles known as acidocalcisomes,
which have some traits in common with vacuoles, for example,
possessing H+-pumping pyrophosphatase in their membranes (Kulaev
et al., 2004).
Electron microscopy in combination with the results of 31P NMR
spectroscopy of living cells can provide a realistic picture of the P distribution within microalgal cells. The aforementioned technique provides
information (though not always comprehensive) on P speciation, compartmentalization, its chemical surrounding, P chain length, formation
of cation complexes, and P exchange (Ginzburg et al., 1988; Martin
et al., 1982; Mitsumori and Ito, 1984). Wood and Clark (1988) compiled
a comprehensive account of polyphosphate study methods.
2.6.4. Physiological roles of polyphosphates
The mechanisms of polyphosphate's biosynthesis and catabolism
and its physiological significance for photosynthetic microorganisms remain largely elusive. Polyphosphate is undoubtedly a P reserve but may
also play a number of other essential roles that remain largely unexplored (Rao et al., 2009). Polyphosphates are secondary products of
photophosphorylation, meaning that they are formed after organic
phosphates (including ATP) and that their turnover rate is slow relative
to that of other P species in the cell, even under conditions of Pi starvation. Thus, polyphosphate may serve as an energy reservoir for ATP formation through (the) polyphosphate kinase enzyme(s), similar to
analogous enzymes identified in bacteria and yeast (Wood and Clark,
1988).
Another suggested function for polyphosphate is regulatory; it is involved in regulation of the synthesis of ATP and other phosphorylated
compounds, acting as a metabolic “buffer” between different cell compartments (Cembella et al., 1982; Kuhl, 1974). The intracellular concentrations of ATP and Pi, as well as the Pi level in the culture medium,
regulate the biosynthesis and degradation of polyphosphate in
microalgae (Cembella et al., 1982).
Purified granules contained polyphosphate complexes with calcium
and magnesium as the most common inorganic components (see
Kulaev et al. (2004) and references therein). X-ray microanalysis of
the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii
showed large amounts of P, magnesium, calcium, and zinc. Immunofluorescence microscopy revealed a vacuolar-type proton pyrophosphatase (H+-PPase) in this compartment. Purification of the electrondense vacuoles using iodixanol density gradients showed preferential
localization of H+-polyphosphatase and H+-ATPase activities in addition to high concentrations of pyrophosphate and polyphosphate
(Ruiz et al., 2001). There are also reports implying that polyphosphate
could be involved in the sequestration of heavy metals in microalgal
cells (see Nishikawa et al. (2006) and references therein). Studies carried out with C. reinhardtii suggested that distinct mechanisms regulate
cell-wall-associated and intracellular polyphosphate, and that the former might protect the cells against toxic compounds or pathogens during cytokinesis, which is when they are more vulnerable (Werner et al.,
2007). The 31P NMR studies carried out in vivo by Pick et al. (1990)
showed that in the unicellular alga Dunaliella salina long-chain
7
polyphosphates are hydrolyzed, generating protons and/or energy
which could facilitate the maintenance of pH homeostasis under condition of alkaline stress.
Phaeodactylum tricornutum responds to hyperosmotic stress by a
sizeable elongation of polyphosphate chains and a decrease in the
total amount of polyphosphate, whereas exposure to hypoosmotic
stress resulted in a higher content of shorter polyphosphate chains
and increased total polyphosphate content. These changes might reflect
the buffer role of polyphosphate and its involvement in adjusting intracellular osmotic pressure and storing elements in an osmotically inactive form (Raven and Knoll, 2010; Werner et al., 2007).
Finally, accumulation of P reserves in the form of polyphosphate
rather than Pi also increases the relative cell density and affects the vertical migration of algae between the aphotic, P-rich, deep-water layers
and photic but P-poor, surface waters (Raven and Knoll, 2010). A high
polyphosphate content, which functions as ballast, may impede the upward migration.
2.7. Strategies of Pi sequestration in algal cultivation systems
Exponential growth of algae is observed when a culture is limited by
neither nutrients nor light (e.g. due to self-shading). Sustained exponential growth can be maintained in turbidostat or chemostat regimes,
leading to a constant uptake of P that is proportional to the stoichiometric concentration of the element in the biomass (Saito et al., 2008). The
growth and P uptake rate are linearly proportional to the biomass and
its P content, respectively. In this scenario, the rate of P uptake is
constrained by cultivation technology and algal biology. On the technological side, the rate of P uptake per unit volume increases with the
number of cells in the culture volume until the energy-requiring uptake
becomes limited by self-shading or by CO2 and O2 mass transfer. For example, a high rate of P uptake per culture volume can be expected in a
thin layer photobioreactor where algae are saturated by light and CO2
under high cell densities, provided they still attain a high growth rate
(Doucha and Lívanský, 2014). The higher the culture density that still allows exponential growth, the higher the P uptake rate attained with the
given cultivation system and algal species is. On the biological side, the
faster the specific growth rate of the alga and the higher the quota (stoichiometric fraction) of P in the algal cells, the higher the P uptake rate is.
The turbidostat or chemostat cultivation systems discussed above
lead to a stable P cell content. However, the cell P stoichiometry can
largely change in dynamic conditions, such as during the overshoot
phenomenon when starved cells are exposed to a high P environment (Herbert, 1961). Eppley and Strickland (1968) concluded that
the growth rate under dynamic environmental conditions is more
closely related to cellular content than to the actual external concentration of nutrients. It was suggested that the kinetics of P uptake depend on both the nutritional status (“nutritional history,” cell P
quota) and the growth rate of the alga (Cembella et al., 1982;
Droop, 1983; Grobbelaar, 2013).
3. Phosphorus uptake under different cultivation conditions
3.1. Orthophosphate concentration
In continuous cultures, such as turbidostats or chemostats, the Pi
concentration in the medium is constant, allowing the identification of
different uptake regimes (Brown and Shilton, 2014) and allowing rigorous pH control to discriminate between the uptake of different ionic
species (Cembella et al., 1982). Water's typical pH range (which encompasses the optimum pH range for many microalgal species) is 5–9, and
–
the dominant free Pi species are HPO2–
4 and H2PO4. Ullrich-Eberius
(1973) postulated that the cell only takes up H2PO–4, which is predominant at a lower pH. Nevertheless, the optimum pH for Pi uptake covers a
wide range, suggesting that HPO2–
4 is also transported. The higher ionic
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charge of HPO2–
4 must be compensated for with a higher ratio of cation
co-transfer to facilitate passage through membranes.
In this context, one should take into account that the pH in wastewater stabilization ponds (WSPs) is highly variable. Thus, vigorous uptake
of nitrate and dissolved inorganic carbon by microalgal cells might shift
the pH to alkaline values, displacing the P species equilibrium and
thereby affecting the bioavailability of the inorganic P dissolved in
wastewater.
With respect to concentration,5 Powell et al. (2008, 2009) demonstrated that in the range of 5–30 mg L−1 P, the luxury uptake by a
microalgal consortium was independent of the P concentration in the
medium. In the lower part of this range, between 5 and 15 mg L−1 P
in the medium, the algae stored polyphosphate primarily in the form
of acid-soluble polyphosphate, whereas storage of acid-insoluble
polyphosphate depot occurred above 15 mg P L-1. This observation
can be tentatively generalized as a threshold below which the luxury
uptake is directed towards acid-soluble polyphosphate (C and A in
Fig. 2), whereas above this threshold concentration, storage goes also
to acid-insoluble polyphosphate (D and B in Fig. 2).
3.2. Photosynthesis, irradiance, and temperature
Phosphorus uptake and storage are energy-requiring processes that,
under autotrophic conditions, require sufficient light to drive photosynthesis. According to the ample evidence reviewed by Cembella et al.
(1982), photophosphorylation, with a significant contribution from
the ATP generated during cyclic electron flow around Photosystem I,
provides a major source of energy for active Pi uptake. The role of cyclic
photophosphorylation in the Pi uptake was supported by the investigation of simultaneous uptake of Pi and nitrate in Ankistrodesmus braunii
(Ullrich-Eberius, 1973).
Interestingly, increasing light intensity may have no significant effect on acid-insoluble polyphosphate and may even induce a decline
in the acid-soluble polyphosphate (Brown and Shilton, 2014). Probably,
the cells that divide more rapidly in high irradiance start to assimilate
acid-soluble polyphosphate for the biosynthesis of cellular constituents
at a higher rate than the polyphosphate replenishment (Powell et al.,
2008).
Kulaev et al. (2004) suggested that photosynthesis is required to
provide the necessary free energy for polyphosphate synthesis, but
the size of the polyphosphate pools is a complex function of
polyphosphate synthesis and hydrolysis, as well of P-dependent metabolic pathways requiring phosphorylation (e.g. biosynthesis of phospholipids, sugar phosphates, and nucleotides). Polyphosphate C is
synthesized only in the light, but polyphosphate B can be formed
using energy reserves regardless of illumination (Fig. 2). Consequently,
the Pi required for RNA synthesis could be obtained directly from external Pi in the light or indirectly from polyphosphate B in the dark.
Polyphosphate D is mobilized in the light for RNA synthesis only in
the absence of external Pi (Miyachi and Miyachi, 1961; Miyachi and
Tamiya, 1961a, 1961b). The complexity of the interactions between
photosynthesis, P uptake, and polyphosphate pools was further revealed by findings that in the chlorophytes A. pyrenoidosa (Senft,
1978) and Pediastrum duplex (Lehman, 1976), the rate of photosynthesis at saturating irradiance depended upon the cell P quota, suggesting a
relationship between Pi uptake and photosynthesis. The relationship
may be circular; increased photosynthetic capacity probably elevates P
demand for anabolic reactions, and the anabolic reactions further stimulate Pi uptake.
The cultivation of algae at a low CO2 level and plentiful irradiance
slowed cell growth and division but increased polyphosphate content
per cell weight or volume, suggesting that the luxury Pi uptake continues under carbon limitation (Ullrich, 1972). This observation further
supports the significance of cyclic and pseudocyclic electron flow for
luxury Pi uptake in microalgae (Allen, 2003; Raven and Glidewell,
1975). With sufficient Pi supply, the energy that cells cannot use for linear photosynthetic energy transfer is probably directed to luxury P uptake (Cembella et al., 1984).
From a biotechnological perspective, this means that the microalgal
culture performing the sequestration of P ought to be optimally illuminated. With high cell density systems (N1 g dry cell weight per L), high
irradiance is provided only to cells that pass near the surface (b 10 mm),
whereas the photon flux densities in deeper and thus shaded suspension layers are not sufficient to drive photosynthesis (Richmond,
2004). On the other extreme, the light ought not to be strong enough
to cause photoinhibition (Richmond, 2004; Zarmi et al., 2013). One
needs to maximize the surface-to-volume ratio of the cultivation system
and optimize the mixing of the suspension. This is the foundation on
which general principles for photobioreactor design have been formulated (Lee et al., 2014; Zarmi et al., 2013; Zittelli et al., 2013). These general principles should be taken into account when developing solar
irradiance-driven microalgae-based systems for P sequestration. Irradiance may also have different effects depending on the suspension temperature. Powell et al. (2009) suggested that increased P uptake at
optimal temperatures is due to an elevated rate of acid-insoluble
polyphosphate formation.
Energy for Pi uptake and polyphosphate synthesis can also be obtained by oxidative phosphorylation, e.g. in the dark or under heterotrophic growth conditions. Under P limitation, this contribution can
outweigh the amount of energy coming from photophosphorylation
in certain organisms such as Euglena or dinoflagellates, but not in
chlorophytes studied (Ch. pyrenoidosa, Scenedesmus sp., Hydrodictyon
africanum; see Cembella et al., 1982, 1984). In summary, the capability
of polyphosphate formation in darkness might be beneficial for the sequestration of P from wastewater enriched with organic pollutants.
4. The potential of microalgal biotechnology for
phosphorus recycling
4.1. Real-world phosphorus sequestration
An overwhelming majority of studies on P uptake by microalgae
have been carried out under controlled conditions in the laboratory
and using artificial growth media or synthetic wastewater. Although
such studies are necessary to obtain a detailed insight into the process
of luxury P uptake, it is the field studies with industrial-scale P sequestration facilities, such as high-rate algal ponds or waste stabilization
ponds (WSPs), that can lead to practical applications. It was shown
only recently that luxury uptake of P by microalgae does indeed regularly occur in full-scale WSPs where the typical P content is in the range
15–30 mg L−1, resulting in 0.21% to 3.85% per volatile suspended solids
dry weight (Brown and Shilton, 2014; Powell et al., 2011b). These figures are consistent with laboratory results supporting the feasibility of
scaling up from laboratory to industry scale (see Brown and Shilton
(2014) and Fig. 5).
For effective P-sequestration on an industrial scale, the P-load rate in
the facility must correspond to the P uptake capacity of the microalgal
culture. In such optimal conditions, Mulbry et al. (2008) reported a
microalgal P removal efficiency of 70% to 90% at a P supply rate of
0.15 g m−2 day−1. An increase in P supply above the P uptake capacity
of the algal cells will not result in an increase of P sequestration.
4.2. Monoculture or association?
5
In this respect, it is also important to remember that a high concentration of Pcontaining compounds does not necessarily imply that all the contained P is bioavailable.
It is even possible that algae are P-starved when P in the medium is in a form that is not
biologically available.
Associations of several microorganisms, including microalgae,
cyanobacteria, and heterotrophic bacteria, have distinct advantages for
environmental applications, including better stability and improved P
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
9
Fig. 5. Summary of the biological P removal mechanisms in waste stabilization ponds. Phosphorus enters waste stabilization ponds as a component of the influent wastewater. The three
main pools of P are shown in bold: in wastewater, biomass, and sludge. The mechanisms responsible for the movement of P between these three pools are identified, and the key
environmental factors that have been shown to influence these processes have been indicated in italics. Adapted from (Brown and Shilton, 2014).
sequestration by increasing bioavailability of P in wastewater
(de-Bashan and Bashan, 2004; Posadas et al., 2014). In many cases,
wastewater contains organic P species that are scarcely available for uptake by eukaryotic microalgae. At the same time, their associated bacteria often possess enzymes to mineralize the organic P (Lim et al., 2007).
Nevertheless, microalgae such as Chlorella are able to hydrolyze and
take up organic P in the form of phytates (Cembella et al., 1982),
which are abundant in waste streams generated by the agricultural,
raw-material processing industry. The extracellular phosphatase responsible for this is likely to enhance the release of Pi from colloidal P
as well (see Fig. 2). Dissolved alkaline phosphatases are reportedly capable of directly degrading particulate matter, resulting in rapid Pi release (Jansson et al., 1988). S. quadricauda, which is incapable of
utilizing phosphomonoesters or pyrophosphate, appeared to overcome
this limitation by associating with bacteria that released Pi from the substrates, which was not directly available to the microalga (Kuenzler,
1965).
The most radical combination of microalgae and bacteria suggested
so far is plant growth-promoting (PGPR) bacteria (e.g. Azospirilla),
used in agriculture to enhance the growth and nutrient removal capacity of microalgae from wastewater (de-Bashan and Bashan, 2004). Furthermore, these artificial associations (thus far not found in nature)
significantly increased the removal of ammonium and soluble Pi ions
compared to the use of microalgae alone (de-Bashan et al., 2002, 2004).
Another benefit of the association of microalgae with heterotrophic
bacteria is a more sustained supply with inorganic carbon (CO2). The atmospheric concentration of CO2 limits microalgal growth, so sparging
the cultures with CO2-enriched gas mixtures generally increases
microalgal growth rate but the cost might be significant (Brennan and
Owende, 2010; Park et al., 2011). This cost can be mitigated, for example, by the use of point sources of CO2, such as flue gas from heat and
power plants, but this requires additional infrastructure that may not
be available. In the case of wastewater treatment, respiration by heterotrophic bacteria normally present in the culture generates additional
CO2.
microalgal isolates because in these P-rich systems natural selection of
the strains with high potential of taking up and storing large quantities
of P in the cell may take place. Another area worth further exploration is
the selective pressure of cycles of P enrichment and starvation, similar
to EBPR (Mino et al., 1998), creating a “feast and famine” regime that
may effectively select for polyphosphate-accumulating algae.
An alternative to using or selecting native organisms is to engineer
strains with higher P uptake and storage capacities. However, according
to current legislation, the use of engineered algal strains is permitted
only in closed cultivation systems such as PBRs (Shilton and Blank,
2012). Whereas P uptake and regulation systems are relatively well
studied in prokaryotes (Keasling et al., 2000) and in higher plants
(Raghothama, 2000; Rausch and Bucher, 2002), our knowledge about
the genes responsible for P uptake and their expression, regulation,
and conservation in algae is scarce, limiting genetic manipulation towards this goal. Nevertheless, the rapid progress in microalgal genomics
(Blaby et al., 2014; Grossman, 2005; Grossman and Aksoy, 2015), metabolomics, and phosphoproteomics opens new opportunities for significant breakthroughs in genetic manipulations towards enhanced
microalgal (luxury) P uptake in the near future.
Possible directions for engineering microalgae for enhanced P uptake include: 1) increasing the rate of Pi transport through membranes;
2) boosting cell storage capacity by promoting the biosynthesis of
polyphosphate; and 3) knocking out the regulation mechanisms
preventing P uptake when the cell is saturated with P. The targets for
manipulation may include genes coordinating the P-starvation response in microalgae. A nuclear gene-regulator, P starvation response
1 (PSR1) of the chlorophyte C. reinhardtii can serve as an example
(Shimogawara et al., 1999; Grossman and Aksoy, 2015). Relevant information can also be obtained from homological structures and processes
in intensely studied prokaryotes (Keasling et al., 2000) and higher
plants (Raghothama, 2000; Rausch and Bucher, 2002). A detailed account of the molecular mechanisms and functional genomics of the Pi
uptake in higher plants and mycorrhiza can be found in excellent reviews by Bucher (2007); Schachtman et al. (1998), and Walder et al.
(2015).
4.3. Providing algal strains for luxury P uptake
4.4. Choosing the cultivation mode for microalgal phosphorus sequestration
It is preferable to select strains capable of rapid growth and
possessing large cell P quota. In a P-eutrophicated environment, cells
with increased abilities for P uptake and incorporation in the cell structures should be selected. This approach seems to be feasible due to high
clonal variability within and between microalgal subpopulations
(Cembella et al., 1982). Accordingly, wastewater treatment plants and
stabilization ponds deserve a closer look as sources of promising
4.4.1. Open ponds
Waste stabilization ponds (WSPs) are shallow man-made basins accumulating wastewater, which is treated over a long time with a combination of aerobic and anaerobic bacteria as well as microalgae (Mara,
1996; Von Sperling, 2007; Shilton and Walmsley, 2008). WSPs are
well established and widespread for the treatment of domestic and
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
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A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
industrial wastewater in regions with ample land and sunshine (Mara,
2004; Von Sperling, 2007). Phosphorus removal in WSP is mainly accomplished by adsorption to sediment and uptake by heterotrophic
and autotrophic microorganisms such as microalgae and cyanobacteria
(Bitton, 1999). WSPs are further divided into anaerobic, facultative, and
maturation/oxidation ponds that are often set up in a series to achieve a
reasonable efficiency of wastewater treatment (Mara, 2004). Microalgae exert a sizeable contribution to P removal in the upper (photic)
layer of facultative ponds which can be primary or secondary (Bitton,
1999; Von Sperling, 2007). In addition, the elevated pH caused by photosynthesis and nitrogen fixation by the microalgae facilitates phosphorus precipitation in form of phosphates (Bitton, 1999).
High-rate algal ponds (HRAPs, Nurdogan and Oswald, 1995) were
the first systems for intensive algal cultivation adapted for wastewater
treatment and involving mixing and CO2 addition. HRAPs further
evolved into the concept of “luxury uptake ponds,” facilitating uptake
of nutrients such as P by microalgal cells from treated wastewater. P uptake rates from wastewater could be increased at least three-fold by
triggering luxury P uptake mechanisms in microalgae (Powell et al.,
2009). The characteristic features of high-rate algal pond and luxury uptake ponds include: 1) relatively high P load rates; 2) low pond depth
(typically b30 cm) and hence high light intensity; and 3) aeration
and/or mixing of the suspension in the pond. Importantly, sustained P
uptake in a high-rate algal pond or a waste stabilization pond requires
a periodic removal of the microalgal biomass, otherwise the P would
simply cycle between the liquid and the sludge layer resulting in minimal net P removal (Powell et al., 2011b).
As discussed by Powell et al. (2009), the luxury uptake pond should
be vertically mixed to ensure uniform illumination of microalgal cells.
After the P-rich biomass is harvested, microalgae leaving the “luxury
uptake pond” would then be harvested and the remaining liquid
would be further treated in the main pond system.
Generally, for efficient recovery of P the treatment process in the
luxury uptake pond should focus on concentrating the maximal
amount of P in a minimum amount of biomass over the shortest possible retention time (i.e., a high P supply rate). Nevertheless, the
complexity of P transformations in the pond environment and fluctuating factors make the development of a luxury uptake process
challenging.
4.4.2. Photobioreactors for suspension cultures
The dominant technology for microalgal P sequestration is based on
the open-pond systems because they are inexpensive and simple to
construct. Closed photobioreactors (PBR) can be considered for special
situations in which, for example, a wastewater effluent is used as an inexpensive nutrient source in emerging large-scale applications, such as
third generation biofuels or CO2 sequestration. Also, PBRs may be considered when high costs of harvesting microalgae from diluted suspensions, water evaporation, dependence on climatic conditions, large
footprint, and unstable yield and biomass quality (Abeliovich, 2004) in
open-pond systems outweigh their lower cost.
Zittelli et al. (2013) recently reviewed the design of low-cost,
energy-efficient PBRs. Vertical, horizontal, tubular, circular, and
flat-panel reactors are the most widespread PBR types. The key factors of successful use of microalgae for sequestration of P from
wastewater are: 1) the choice of rapidly-growing strains that are tolerant to eutrophic conditions and a particular wastewater composition; 2) an energy-efficient PBR design supporting intensive
cultivation of microalgae; and 3) fine-tuning of the illumination intensity, mixing rate, and temperature based on a deep understanding of the physiology of luxury P uptake by microalgal cells. For
detailed account of specific advantages and disadvantages of different PBR designs, we refer the reader to recent reviews (Lee et al.,
2014; Zittelli et al., 2013). Microalgae grown in PBRs possess a high
potential for P removal (N90%; Shilton et al., 2012; Posadas et al.,
2014) and PBR allows, as a closed system, the use of engineered
algal strains, so we believe that the application of the closed cultivation systems holds promise for P recovery from wastewater, especially in regions with temperate and cold climates or high land cost.
4.4.3. Immobilized microalgae
An interesting alternative to suspension cultivation is the use of
immobilized microalgal cells (de-Bashan and Bashan, 2010).
Immobilized-algae PBR designs include fluidized- and packed-bed,
parallel plane, hollow fiber constructions; for efficiency comparisons, see Mallick (2002). The advantages of immobilized algae cells
include steady growth, lack of mixing requirement, high waterpurification efficiency, simplified biomass harvesting, and increased
cell retention (reduced wash-out) (Boelee et al., 2011; Mallick,
2002). Cells are usually immobilized in alginate gels (Mallick,
2002) or form biofilms themselves (Boelee et al., 2011). The cells of
Chlorella vulgaris immobilized in alginate granules removed up to
70% of P from wastewater (Travieso et al., 1992). The cells of
Scenedesmus immobilized in alginate sheets also efficiently removed
nutrients from tertiary wastewater (Zhang et al., 2008). Thus, algalturf scrubbers (ATS) used for farm waste treatment support the load
rate of 2700 kg of N and 400 kg of P per 1 ha per year, yielding
27,000 kg ha− 1 of dry microalgal biomass (Pizarro et al., 2006). A significant advantage of this technique is that the turf with P-enriched
algal biomass immobilized in it will be an almost ready-made P
bio-fertilizer. Nevertheless, the main problems with this method
are nutrient limitation by diffusion, photoinhibition, a suitable immobilization method, and biomass harvesting. Immobilized algae
also offer the possibility to regenerate the P uptake capacity of the
immobilized microalgae through P starvation (Kaya and Picard,
1996).
4.4.4. Thin-layer cultivation systems
As argued above, the systems with a short light path across the algal
suspension represent a significant advantage because they can support
much higher cell densities taking into account that the light absorption
is governed by light path (layer thickness) and culture density. The
same areal density in a 5-mm, thin-layer system as compared to a 30cm-deep pond means about the same light capture efficiency but with
a volumetric density that is 60 times higher in the thin layer. A higher
volumetric density means proportionally lower harvesting costs and
lower costs of pumping or transporting. Because the surface-tovolume ratio of such a thin-layer cultivation system is 60 times higher,
the mass transfer of CO2 and O2 is proportionally better in the thin
layer. Also assisting the mass transfer, the vertical flow gradient in the
thin-layer system is high, thus promoting efficient mixing of the suspension close to the liquid surface. The high surface-to-volume ratio, effective mixing, short light pathway, and high culture density in the late
exponential phase are typical for thin-layer systems (Doucha and
Lívanský, 2014). At Forschungszentrum Jülich's AUFWIND facility, O.
Pulz of IGV GmbH has recently constructed an interesting modification
of this approach, in which the algal suspension is sprayed in droplets
over a system of multiple stacked horizontal nets (manuscript in
preparation).
4.5. Production and use of the phosphorus rich microalgal biomass
4.5.1. Production of phosphorus rich algal biomass using waste streams
Microalgae can be grown in wastewater or other liquid waste
streams that are rich in nutrients, including phosphorus (Park et al.,
2011; Cabanelas et al., 2013; Ray et al., 2013). Åkerström et al. (2014)
described how residual P and N in sludge liquor resulting from a
solid–liquid separation of digestate could be a suitable medium for Chlorella spp. biomass production. The production of algae biomass to recycle and recover nutrients from manure, as well as the treatment of
manure effluents for algae biomass production has been demonstrated
successfully (Mulbry et al., 2005, 2008; Wilkie and Mulbry, 2002).
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
Anaga and Abu (1996) reported the efficient removal of P and N from
the effluent of a fertilizer company using Chlorella and Spirulina, demonstrating both effluent purification and biomass production for further
applications (Cho et al., 2013). In this way, algae biomass production
can become an integral part of treating and re-cycling the wastewater
streams (Cabanelas et al., 2013).
4.5.2. High-value P-rich products
The P-rich biomass of microalgae has a number of different uses.
Kulaev et al. (2004) reviewed present applications of polyphosphate.
In particular, P-rich microalgal biomass is considered to be a potential
source of polyphosphate. Polyphosphates are in high demand in different areas of medicine as well as in the biomaterials and food industry.
Medical usage of polyphosphate is defined by its antiseptic, cytoprotective, and antiviral activities. At a concentration of 0.1% or higher,
polyphosphate exerts a bactericidal and bacteriostatic effect. Interestingly, polyphosphate with more than four Pi residues inhibited human
immunodeficiency virus type 1 (HIV-1) infection of cells in vitro, presumably by inhibiting adsorption of the virus on cell surface (Lorenz
et al., 1997). Polyphosphates are used in the synthesis of new biocompatible materials such as Ca-polyphosphate bioceramics and scaffolds
for tissue regeneration (Waldman et al., 2002). The food industry is
also an important consumer of polyphosphates, which are used as multifunctional ingredients (E-451 and E-452) in food, such as ham, bacon,
meat poultry, fish, and shellfish due to their high buffering capacity as
well as sequestering and antibacterial effects. At the same time, cultivation of microalgae in wastewater may preclude its use for high valueadded products such as food additives or medical applications. In this
context, one should also consider that the high value products are typically niche market products. The algal biomass produced on waste
streams is typically much larger than the demands from these markets
and media costs are rarely an issue there.
Therefore, microalgae grown on waste streams are typically considered for low-cost, high volume biofuel production (Park et al., 2011). In
this context, one can foresee the P-rich residue that remains after the
biofuel lipid extraction being used as a bio-fertilizer or as a source of
polyphosphate. In another large volume approach, algal biomass was
used as a co-ferment, increasing the yield of methane (Schwede et al.,
2013). The integration of microalgae production with anaerobic fermentation for biogas generation and the further processing of the
resulting digestates for bio-fertilizers opens research avenues for producing added-value products and closing nutrient loops (Uggetti et al.,
2014). The use of pure digestates as bio-fertilizers and for soil conditioning purposes comprises numerous benefits for plant production (Möller
and Müller, 2012).
4.5.3. Algal bio-fertilizer
The use of algal biomass grown on P-rich waste streams for biofertilizer production occurs on a scale where supply and demand can
be balanced (Fig. 4). As reviewed by Metting (1996), some rice growers
are currently using cyanobacteria rather than eukaryotic algae as a biofertilizer. Cyanobacteria's ability to fix nitrogen from the atmosphere
comprises a significant added value. Cyanobacteria applied as living
cells and pretreatment of the plant seeds with cyanobacterial inocula
led to enhanced germination rates of numerous species. This effect is
likely due to phytohormonal and enzymatic activities that together
with nutrients promoted plant performance (Adam, 1999). Application
of living algal suspensions may, however, be hampered by difficult biomass conservation, implying that more manageable forms of algal biomass processing, logistics, and application must be found.
To assess the competitiveness of P-rich microalgal biomass versus
traditional synthetic P fertilizers as well as their compatibility, one
must critically compare the biomass with direct application of digestate
or raw manure to soils (Laboski and Lamb, 2003). Mulbry et al. (2005)
published one of the few studies evaluating the fertilizer value of
dried algal biomass grown on anaerobically digested dairy manure.
11
They found that for two soil types, levels of Mehlich-3 extractable P
(available to plants) rose with increasing levels of algal amendment.
However, the P levels in the soil before the application also influenced
the positive effect. As described in the study by Mulbry and fellow researchers, plant growth experiments showed that 20-day-old cucumber
and corn seedlings grown in a potting mix containing algae assimilated
38% to 60% of the P applied with the microalgal biomass. The plants
grown in algae-amended potting mixes were equivalent to those
grown with comparable levels of fertilizer-amended potting mixes
with respect to seedling dry weight and nutrient content. An added benefit of algal biomass is that it does not need to be tilled into soil, which is
necessary for mineral P fertilizers. The algal biomass could be sidedressed into growing crops, thereby saving much labor and energy. According to the estimations by Mulbry et al. (2005), the amount of algal
biomass grown on the manure from a 100-cow dairy farm could support 4 ha of production, and at these application rates (6.5–10 t biomass
ha−1), loading of heavy metals from the algal biomass would be well
below the permitted level (Mulbry et al., 2005). Also, Powell et al.
(2011b) showed that sludge from waste stabilization ponds used for
wastewater treatment with microorganism consortia including
microalgae has potential as a bio-fertilizer. The presence of pollutants
such as pathogens and micropollutants may be a point of concern. However, the risks associated with the use of bio-fertilizers in agriculture are
typically low (Mulbry et al., 2005; Wilfert et al., 2015). Even when a material is technically safe, legal barriers may prohibit such a product from
entering the market. For example, the European Fertilizer Regulation
(EC2003/2003) contains a list of materials that are allowed as fertilizer,
mainly single (mineral) compounds. Currently, a revision is being made
towards a more functional classification, including specifications on
minimal nutrient levels and maximum levels of undesired compounds.
Finally, and probably most importantly, the public perception on safety
of bio-fertilizers may hinder market introduction. This hindrance can
only be eliminated by the careful monitoring of the possible sources of
concern and by presenting this information in a transparent manner.
The microalgae that settle into waste-stabilization pond sludge contain significant quantities of polyphosphate (up to 71% of the total P in
the sludge). At 25 °C, the sludge releases P from the polyphosphate at
a rate of 4.3–12.4 mg P d− 1 per gram of total suspended solids. The
rate of P released from fresh microalgal sludge grown under laboratory
conditions was even higher, comprising 160 mg P d−1 per gram of total
suspended solids. It is noteworthy that after the initial release phase, P
was re-assimilated in this system and some polyphosphate was reformed, likely due to bacterial growth in the sludge. The microbial
breakdown of organically bound P into inorganic forms available for
algae growth has previously been reported (Zhao et al., 2012). The release of plant-available Pi from polyphosphate accumulated in the biomass depends on soil microbial phosphatase activity, among other
factors. Generally, this process is slow; therefore, it possesses a high potential of supplying P at a rate just sufficient for crops, reducing the
probability of oversupply and waste that is characteristic of mineral P
fertilizers (Ray et al., 2013). Ray et al. (2013) carried out a preliminary
comparison of P release by chemical superphosphate fertilizers and
the equivalent amount of P-rich microalgal biomass applied to Pdeficient soil for rice seedlings. The amount of plant-available P released
by the chemical fertilizer was eight times higher than the amount that
the crop plants could assimilate. In contrast, in case of the microalgalbased bio-fertilizer, the released plant-available P was only 3–4.4
times higher, suggesting a more sustainable and environmentally
friendly crop fertilizer.
5. Conclusions and outlook
The reliance of humanity on the rapidly depleting, non-renewable
phosphate rock is a grave, though currently underestimated threat to
food security. Continued wasteful use of phosphorus from phosphate
rock also leads to a permanent excess of P in the environment, which
Please cite this article as: Solovchenko, A., et al., Phosphorus from wastewater to crops: An alternative path involving microalgae, Biotechnol Adv
(2015), http://dx.doi.org/10.1016/j.biotechadv.2016.01.002
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A. Solovchenko et al. / Biotechnology Advances xxx (2015) xxx–xxx
disturbs natural processes and human health. Efficient recovery and
reuse of P from waste streams are central for sustainable food security
and a healthy environment.
Microalgae have a significant potential for P recycling, particularly
via algae-based bio-fertilizers produced on P-rich waste streams.
Algae can perform sustained luxury P uptake driven by sunlight, grow
fast, use nutrients available in wastewater, and form biomass suitable
for bio-fertilizer production. Positive synergistic effects can be achieved
by coupling wastewater treatment with bio-fertilizer and biofuel production. Algae assimilate CO2, which is among the most abundant and
noxious greenhouse gases.
To turn this potential into a widely used cycle going from waste
streams to crop plants, further research is needed on P uptake mechanisms, their regulation, and their integration in metabolic networks of
algae. The research on P transport and transformation pathways may involve advanced “omics” techniques to identify target genes and enzymes. The new knowledge generated may enable the engineering of
strains with enhanced capability of luxury P uptake. Simultaneously,
this will be supported with bioprospecting native microalgal strains
and associations that are capable of rapid sustained uptake of P and
withstanding the fluctuating background of other pollutants in realworld wastewaters. Further optimization of the current cultivation systems will occur in parallel with efforts towards the development of new
low-footprint platforms for sequestration of P from wastewater. The
cultivation conditions leading to the most effective accumulation of P
in the algal biomass must be identified, as well as optimal harvesting
procedures. A further objective is to identify the form of algal biomass
that is best suited for application as a fertilizer in view of logistics, stability, application approach, and delay of the phosphorus release. This will,
for example, include investigation of P availability and potential adverse
effects of surface-applied algae suspensions, such as clogging the soil
surface or soil aggregate formation, potential decline in surface water
absorption or nutrient access for the crops. A better understanding is required about the interaction of algal biomass with the soil microbial
community, particularly with bacteria and mycorrhiza (Malik et al.,
2012), including detailed mapping of the phosphorus flux from algal
biomass to soil and plant roots. The pathways of phosphorus release
from algal cell walls (Sanudo-Wilhelmy et al., 2004; Xu et al., 2014)
versus intracellular phosphorus pools require further differentiation. Direct effects of phytohormones such as auxins, cytokinins, abscisic acid,
and ethylene contained in algae on plants (Riahi et al., 2013) also
need further attention. Of separate concern are allelochemicals and/or
cyanotoxins which effects are likely plant-dependent and need further
investigation (Berry et al., 2008). Comprehensive studies of the effects
of algae-based bio-fertilizers on various crops and arable soils remain
essential.
Acknowledgments
The authors are grateful to Johan Grobbelaar and Leon Korving and
three anonymous reviewers for their critical reading and many valuable
comments that helped to improve the manuscript. We apologize to the
colleagues whose papers we could not directly cite because of space limitations. The collaboration in Russia was supported by Russian Scientific
Foundation (project #14-50-00029). AES (BioSC visiting scientist) as
well as LN and NDJ (BioSC project AlgalFertilizer) were financially supported by the Ministry of Innovation, Science and Research within the
framework of the NRW Strategieprojekt BioSC (No. 313/323‐400‐002
13). Financial support of the networking from Russian Foundation for
Basic Research (project #15-34-20096) are gratefully acknowledged.
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