Abstract

Introduction

During the last 50 years, from 1971 to 2021, the population of the earth has increased twofold from ca. 3.9 billion to 7.8 billion, accompanied by a huge demand for energy to satisfy their requirements. Fossil fuels (gas, coal, petroleum oil) and nuclear fission have long been utilized to supply this energy, but their use is now in disfavour due to environmental and health concerns. For instance, combustion of fossil fuels is one of the main factors contributing to global warming (see Pena-Castro et al. 2017). Biofuels derived from plants are sustainable alternatives for fulfilling contemporary demands for energy (Avni and Blazquez 2011; Chen and Zhang 2015), and an important goal of modern plant science is to increase the production of crops suitable for use as renewable energy.

Contemporary “energy plants” include oilseed crops (e.g., rapeseed, Barbados nut, Chinese pistache, yellowhorn) and several microalgae that are used for biodiesel production (Salvi and Panwar 2012; Rahul et al. 2020). The energy basis of other plants is starch (e.g., maize, rice, wheat, cassava), sugars (e.g., sugar cane, sugar beets) or lignocellulose (e.g., Chinese silvergrass, switchgrass, poplar and bamboo species) (Li et al 2010; Xie and Peng 2011; Chen and Zhang 2015). Lignocellulose is very abundant but difficult to process, so that its use in the production of biofuel is challenging (Xie and Peng 2011). Starch- and sugar-containing plant material can be used for both energy generation and food, a potential that may, however, be subject to interest-based restrictions. In China, for instance, starch- and sugar-based biofuel production is strictly regulated due to concerns about securing food sources (Xie and Peng 2011).

Aquatic plants, or macrophytes, have an important advantage over terrestrial crop plants in supplying plant material for producing biofuel in that they do not compete with other crop plants for arable land. Duckweeds are a family of aquatic plants with considerable potential for supplying starch as a feedstock for energy generation. These small floating macrophytes (at most 15 mm in width/length) inhabit the surfaces of still or slowly moving water. Their buoyant bodies or fronds are often flattened ovoids, but can also be globoid, cylindrical, ellipsoidal or linear structures as little as 0.2 mm in width or 0.4 mm in length (see Fig. ​Fig.1).1). They represent a fusion of leaves and stems that may or may not bear hairless ventral roots, and thus constitute the extreme reduction of a vascular plant (Landolt 1986; Acosta et al. 2021). The duckweeds make up the family Lemnaceae that includes 5 genera (Spirodela, Landoltia, Lemna, Wolffiella, Wolffia; see Fig. ​Fig.1)1) encompassing 36 species that are distributed widely around the globe and are characterized by a high diversity of clones or strains that have evolved in response to particular microenvironments (Bog et al. 2020). They are useful for wastewater remediation, as models for physiological, ecological and biotechnological studies, and as standardized ecotoxicity indicator organisms (Ziegler et al. 2016, 2017).

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Fig. 1

Representatives of all five genera of the family Lemnaceae. A Frond of Spirodela polyrhiza 9509 (Germany) forming a turion (arrow); B colony of Landoltia punctata 9589 (India); C colony of Lemna aequinoctialis 9648 (India); D colony of Wolffiella gladiata 8350 (USA). Single fronds are sabre-shaped and pointed toward the tips; E Wolffia globosa 5514 (Thailand). Plants were cultivated in nutrient medium N at 25 °C and 100 µmol/m²/s continuous white light

Although they are flowering plants, most duckweeds flower rarely and reproduce predominantly in the vegetative mode. This generally takes the form of the budding off of new fronds from pouches in the mother fronds, and can also include the formation of overwintering propagules, or turions, from the shoot primordia (Landolt 1986; Landolt and Kandeler 1987; Acosta et al. 2021) upon the impact of appropriate environmental signals (Appenroth 2002). The vegetative reproduction of the fronds can take place very rapidly on account of the high growth potential of these macrophytes (Ziegler et al. 2015; Sree et al. 2015c). The vigorous growth of duckweed fronds is favoured by a high proportion of photosynthetic tissue in the fronds, good exposure to light ensured by the floating growth mode and a proficient uptake of nutrients, and is the basis for the large amounts of biomass that the fronds can rapidly produce. When duckweeds are grown under non-limiting conditions, they possess ample protein, and can be used for nutrition (Xiao et al 2013; Appenroth et al 2017, 2018). Under growth-limiting conditions, often in the presence of any stressor, they contain less protein but often high contents of starch (Xiao et al. 2013; Sree and Appenroth 2014; Cui and Cheng 2015) suitable for producing biofuel. The rapid growth and starch accumulating potential of duckweeds can lead to the production of large amounts of readily renewable biofuel feedstock upon extensive cultivation of the macrophytes (Liu et al. 2021). Duckweeds can be easily collected for processing, and their component starch is easily accessible from their low-fibre, simply constructed tissues (Ziegler et al. 2017). These attributes, together with the lack of encroachment on arable land, make duckweeds a promising vehicle for the sustainable production and exploitation of starch for biofuel energy generation.

In the present review we examine starch metabolism in the fronds of duckweeds from the viewpoints of how growth alone can lead to large-scale starch production and how the intrinsic starch contents of the fronds can be enhanced by manipulation of the growth conditions and by the application of exogenously effector substances. Our aim thereby is to promote the identification of duckweeds well suited for starch production and to determine how to best effect starch accumulation within them for the benefit of biofuel feedstock production.

In deviation from the usual practice in taxonomic naming, “La.” and “Le.” are used in the text as abbreviations for the genera Landoltia and Lemna in order to avoid confusion from two generic names commencing with “L” (cf. Sree et al. 2015b).

Starch accumulation

Green plants use light energy during photosynthesis to drive the fixation and reduction of atmospheric CO₂ to form carbohydrates (photosynthate: initially triose phosphates) in chloroplasts. Photosynthate can be consumed or stored in the photosynthetic tissues themselves, or transported to other parts of the plant to enable synthesis and energy metabolism to proceed there (Stitt and Zeeman 2012). Since duckweed fronds consist mainly of photosynthetic tissue, photosynthate will be either used for the production of new fronds or stored in the frond chloroplasts.

Some of the triose phosphate formed in the light is quite generally stored in the chloroplasts of photosynthetic tissues as starch. This starch is generally degraded during the dark period to its component monosaccharides, which are utilized to maintain synthesis and energy metabolism in the absence of light (Stitt and Zeeman 2012). When growth and development processes consume all (or most) of the photosynthate produced during the light, this “transitory” starch will not accumulate to any great extent. When the production of photosynthate consistently exceeds the demand for it for growth and development, however, starch will accumulate, i.e., increase in content, in the photosynthetic tissues. The starch content of duckweed fronds will accordingly be at a high level when the photosynthesis of the fronds consistently outstrips the use of photosynthate for new frond growth, and the fronds can be made to accumulate starch by imposing conditions on them that either stimulate photosynthesis more than growth or impede growth more than photosynthesis.

Some duckweeds can form overwintering propagules, or turions, instead of normal vegetative fronds upon appropriate environmental signals (Landolt 1986; Landolt and Kandeler 1987). These propagules contain very high levels of starch that are reminiscent of and have the same function as the high starch contents that accumulate in storage and propagative organs such as the tubers of potato and the seeds of wheat and maize. This “reserve” carbohydrate is often stored for long periods during the dormant phases of the mature propagative organs, and then rapidly and completely broken down when the propagative organs germinate and sprout (Zeeman et al. 2010). Turion starch derives from the export of photosynthate from the fronds upon a switch in the developmental scheme of the duckweed from normal vegetative growth to the production of survival organs (Appenroth 2002). The accumulation of turion starch can thus be effected by initiating turion development in the duckweed.

The starch of terrestrial “energy plants” targeted for energy generation is the “reserve” starch of propagative organs of these plants, and the chloroplastic starch of terrestrial crop photosynthetic tissues has not been considered for its exploitable energetic potential. In the following it will be discussed that the “reserve” starch of duckweed turions has only limited potential as a biofuel feedstock, and that duckweed fronds can produce large amounts of chloroplastic starch during the course of their normal growth and can further accumulate starch in their tissues when subjected to the influence of growth regulators, heavy metals, nutrient deficiency and salt stress that influence the relationship between photosynthate production and photosynthate utilization as growth.

Frond starch accumulation due to growth

Varying amounts of starch are found in the frond tissues of duckweeds: starch contents ranging from 3 to 50% of the duckweed dry weight (DW) have been reported (see Landolt and Kandeler 1987). In some more recent studies, control fronds of Landoltia punctata contained starch at approximately 3–5% of DW (Liu et al. 2015a, 2019), those of La. punctata, Lemna aequinoctialis and Wolffia arrhiza at about 10% (Sembada and Faizal 2019) and those of Spirodela polyrhiza at about 20% of DW (Wang et al. 2017). These values are indicative of starch contents that can be maintained in fronds upon cultivation of duckweeds in mineral salt media in the absence of any inhibitory growth conditions or exogenous effector substances (the “intrinsic” starch contents of the fronds). If the cultivation takes place under non-limiting conditions and the intrinsic starch contents remain constant, the duckweeds will accumulate starch in their biomass according to their intrinsic growth potential. Duckweeds are potentially the most rapidly growing of all higher plants, exhibiting relative growth rates of 0.15–0.52 day⁻¹, doubling times of 1.3–4.5 days and relative weekly yields of 2.9–37.8 week⁻¹ under standardized cultivation conditions (Ziegler et al. 2015): the highest growth rate having been recorded for any angiosperm is the 0.69 day⁻¹ reported for Wolffia microscopica 2005 under standardized, but not yet optimized, conditions (Sree et al. 2015c). The large-scale cultivation of high-starch duckweed fronds can thus lead to the accumulation of large amounts of starch for industrial utilization under standardized basic growth conditions without requiring any further measures to enhance starch production. On the other hand, starch contents can vary considerably in the fronds of the various clones or strains that derive from discrete species growing in different microclimates. For example, the starch contents of 9 clones of Le. aequinoctialis growing under identical conditions varied between 2.5 and 22.5% of the tissue fresh weight (Barbosa Neto et al. 2019). The investigation of 16 strains of 6 duckweed species under optimal growth conditions revealed that two strains of Lemna gibba exhibited almost 40% of the frond DW as starch (Sree et al. 2015a), and the mutant sub-1 derived from the Le. aequinoctialis clone 6002 has recently been reported to accumulate starch at>45% of the frond DW under such conditions (Liu et al. 2020). Systematic screening of duckweed clones to identify those exhibiting particularly high starch contents in their biomass is thus indispensable for optimizing starch yields for exploitation (see Ziegler et al. 2017; Sree et al. 2015c).

While large-scale cultivation of high-starch duckweed clones under optimized mineral salt medium and climatic conditions is promising in itself for starch production on an industrial scale, it may be complemented considerably by the accumulation of starch yields in the frond tissues elicited by manipulations of the growth conditions and/or the addition of effector substances to the culture medium.

Phytohormone and growth regulator stimulation of starch accumulation in duckweeds

Phytohormones are plant-endogenous signal molecules that regulate all aspects of plant growth and development. Duckweed fronds contain many of these: Chmur et al. (2020) recently detected eight brassinosteroids and seven free bases and eight conjugates of cytokinins in addition to abscisic acid (ABA) and the gibberellic acid in W. arrhiza. Synthetic growth regulators also influence duckweed growth and development. The phytohormone ABA and especially the synthetic growth retardants uniconazole and maleic hydrazide can be used to enhance the starch content of duckweed fronds over and above the intrinsic starch levels of the tissues; ABA can also induce the accumulation of starch in duckweed turions.

The effects of heavy metals

The application of several (perhaps all) heavy metals to duckweed cultures has two obvious effects: the inhibition of growth (Naumann et al. 2007), and the accumulation of starch in a dose-dependent manner. Some quantitative data for the accumulation of starch in duckweeds subsequent to heavy metal treatment are shown in Table ​Table1.1. In some cases, the starch content attains values of about 50% of DW.

Table 1

Examples of starch accumulation in duckweeds under the influence of heavy metals

Heavy metal	Species, clone	Concentration/time in days (d)	Starch content (% DW)	Reference
Chromate	S. polyrhiza 9500	50 µM/2 d	24	Appenroth et al. (2003)
Cadmium	Le. minor 9441	10 µM/4 d	50	Sree and Appenroth (2014)
Cadmium	30 clones of S. polyrhiza	1 µM/7 d	small effects	Chen et al. (2020a, b)
Cobalt	Le. minor 9441	100 µM/4 d	40.5	Sree et al. (2015b)
Cobalt	La. punctata 0202	8.5 µM/10 d	53.3	Guo et al. (2017)
Lead	Le. gibba	0.25–1.5 mM/13 d	-50% decrease	Sobrino et al. (2010)
Mercury	Le. gibba	40 µM/7 d	27	Yang et al. (2018)
	Le. minor		38
	S. polyrhiza		45
Nickel	Le. minor 9441	≤30 µM/7 d	28.4	Appenroth et al. (2010) and Xylander et al. (1993)
Nickel	S. polyrhiza 9500	≤30 µM/7 d	44.8	Appenroth et al. (2010)
Nickel	La. punctata 0202	8.5 µM/10 d	42	Guo et al. (2017)
Nickel	La. punctata, not specified	50 µM/10 d	ca. 26 (1.5% fresh weight)	Shao et al. (2020)
Selenite (chalcogen)	La. punctata 7449	80 µM/6 d	23.6	Zhong et al. (2016)

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Application of chromate to S. polyrhiza, clone 9500 (old clone term: SJ) resulted in an inhibition of growth and in enhanced accumulation of starch as shown by chemical analysis and transmission electron microscopy (Appenroth et al. 2003). The dose-dependent effects were categorized into four classes. In class 1 (low concentrations of chromate, such as 50 µM for some days or 1 mM for 1 day), inhibition of growth was weak but detectable, and the starch content of the chloroplasts increased. In classes 2–4 (increasing doses of chromate) multiple toxic effects were observed (e.g. reduced PSII core complexes, altered shape of the thylakoids, damaged membrane systems), and the chloroplast starch content was much lower than in class 1. It was concluded that accumulation of starch was enhanced in the presence of low concentrations of chromate, but not at higher concentrations at which the photosynthetic apparatus was already seriously disturbed. A correlation between inhibition of the growth of Le. minor 9441 (old clone term: St) and induction of starch accumulation by chromate was also suggested by Reale et al. (2016), but the starch content was only evaluated on the basis of stained cross sections of fronds, and no quantitative data were available.

Transitions of chloroplasts to chloro-amyloplasts and amylo-chloroplasts, but not to gerontoplasts, were observed upon application of Ni²⁺ to S. polyrhiza 9500 and Le. minor 9441. These morphological transitions were associated with the accumulation of starch in both species as quantified by chemical analysis (Appenroth et al. 2010). The dose–response curves were bell-shaped for both duckweed species, with starch contents increasing at Ni²⁺ concentrations of up to 30 µM and decreasing at higher concentrations. This response was accompanied by increasing inhibition of growth upon increasing Ni²⁺ concentrations. The following conclusions were drawn (Appenroth et al. 2010): a slight inhibition of growth requires the use of less photosynthetically produced photosynthate for metabolism, and the surplus is stored as starch in the plastids as reflected by the rising slope of the bell-shaped dose–response curve at lower Ni²⁺ concentrations. At higher Ni²⁺ concentrations, toxic effects more seriously impair the efficiency of photosynthesis. This leads to the decreasing starch content reflected in the declining slope of the dose–response curves at higher Ni²⁺ concentrations. Supporting results for this interpretation were obtained by the application of Co²⁺ to Le. minor 9441 (Sree et al. 2015b). The starch content of the fronds of this species increased at Co²⁺ concentrations of up to 100 µM, and thus no bell-shaped dose–response curve was observed. However, the increase in the starch content was transient, and decreased at higher Co²⁺ concentrations and upon longer periods of treatment. These results may be interpreted in a manner very similar to that respective of Ni²⁺: longer times of treatment might damage the photosynthesis apparatus to an extent that rendered it unable to supply sufficient carbohydrates for normal metabolism. Photosynthetic efficiency and pigment contents were almost unaffected during the first 4 days (the period of starch accumulation), but seriously impaired thereafter when the starch content decreased (Sree et al. 2015a).

Application of 1 µM Cd²⁺ to 30 different clones of S. polyrhiza from southern China inhibited growth only slightly and resulted in the accumulation of only very low levels of starch (Chen et al. 2020a, b). These authors used the N medium according to Appenroth et al. (1996), which contains 25 µM EDTA. This EDTA concentration will chelate the low concentration of the heavy metal very effectively, preventing the uptake and the effects of the Cd²⁺ (Srivastava and Appenroth 1995). For investigations of the effects of heavy metals on duckweeds, a modified Steinberg medium having an EDTA concentration as low as 4.03 µM is recommended (Naumann et al. 2007).

An unexpected response of duckweeds to selenite was observed upon the application of the chalcogen to one clone of La. punctata (Zhong et al. 2016). At low selenite concentrations (20 µM) growth was not inhibited, but rather slightly stimulated. Nevertheless, the control starch content of 9.2±1.0% of DW increased to 17.5±2.8% of DW after 6 days of treatment with 20 µM selenite, as confirmed by electron microscopy. Thus, instead of inhibiting growth, selenite appears to act in a different manner that results in starch accumulation. It is noteworthy that the authors described the origin of the La. punctata clone as Anhiu province, China, but used the clone number 7449, which indicated a clone taken from Delhi, India. Silver nanoparticle-inhibited growth also stimulated the accumulation of starch in a local clone of La. punctata from Brazil, but the authors evaluated only stained cross sections of fronds (Lalau et al. 2020).

The most important starch synthesizing enzymes are SSS and ADP-glucose pyrophosphorylase (AGP), which consists of three large (APL1, APL2, APL3) and two small subunits (APS). It is generally assumed that the APLs respond to allosteric regulators, whereas the APS act catalytically (Georgelis et al. 2007). Shao et al. (2020) demonstrated in a local strain of La. punctata treated with Ni²⁺ that the mRNA of SSS increased only after treatments of more than 6 h. The regulatory subunit APL3 and the catalytic subunits APS, however, responded to the treatment within two hours, and did so much more strongly than did SSS. The starch synthesizing pathway accordingly mediated the accumulation of starch, whereas transcripts related to starch degradation hardly responded at all to the Ni²⁺ treatment. Further results published by Guo et al. (2017) demonstrated effects of Ni²⁺ and Co²⁺ on La. punctata 0202. These authors investigated the influence of these two heavy metals on the activities of AGP and SSS. Especially AGP responded strongly to 8.5 µM with an increase in activity. In summary, the enhancement of starch accumulation in La. punctata by treatment with Ni²⁺ and Co²⁺ is associated with increased gene expression and enhanced enzymatic activity of AGP and SSS.

When large amounts of starch are required for biotechnological purposes, a two-step procedure has been suggested for using Cd²⁺ and other stressors to effect starch accumulation (Sree and Appenroth 2014). In the first step, plants should be cultivated under optimal growth conditions in order to produce high amounts of biomass. In the second step, stressors should be applied to increase the starch content of the biomass. If Cd²⁺ is to be employed in this regard, for instance, 10 µM of the heavy metal for four days would be effective. However, such practices are fraught with environmental concerns regarding the use of large amounts of heavy metals.

Nutrient stress and starch accumulation

Nutrient stress means mainly limitation or lack of some or even all nutrients in the cultivation medium. As a consequence, this treatment induced stress to the plants and one of the responses is the accumulation of starch. Several studies on the effect of nutrient limitation on duckweeds, for some reasons, have concentrated on only a few species of duckweeds that mostly include S. polyrhiza (Ma et al. 2018), La. punctata (Zhao et al. 2015), Le. aequinoctialis (Yu et al. 2014; Ma et al. 2018) and Le. minor (Sree and Appenroth 2014). The exposure of S. polyrhiza to well water induced starch accumulation up to 26.6% (Cui et al. 2011; Cui and Cheng 2015), that of Le. aequinoctialis to tap water induced 35% starch accumulation (Ma et al. 2018) and that of La. punctata to various nutrient limitation conditions induced starch content up to 30% (Kruger et al. 2020), 45% (Zhao et al. 2015) and 52% (Guo et al. 2020) although at varying time points. Long-term growth in batch culture without replenishing the nutrients also results in nutrient stress, decrease of growth rates and accumulation of starch. This was demonstrated for La. punctata 5632 cultivated in Murashige-Skoog-Medium and Hoagland´s medium. The highest value was reached on day 28 with 4.01% starch per fresh weight (Kiitiwongwattana 2019). Neto et al. (2019) investigated nine clones of Le. aequinoctialis from Brazil for starch accumulation during cultivation in modified Schenk-Hildebrandt media containing 9 mM glucose beside thiamine, pyridoxine, isonicotinic acid and glycine. Under these mixotrophic conditions they observed a remarkable intraspecific variation in the starch content per fresh weight.

Interestingly, the increase of starch content in duckweeds under phosphate limiting conditions was observed as early as 1970 by Reid and Bieleski whilst working with La. punctata (synonym Spirodela oligorrhiza). They reported an increase from 19% per DW in the controls to 29% under phosphate limitation and autotrophic conditions. Under mixotrophic conditions, the starch content increased to ca. 75% but addition of glucose or sucrose to the nutrient medium in order to accumulate starch has no relevance in biotechnology. Almost two decades later, the mechanism behind this increased starch content under nitrate and phosphate limiting conditions was deciphered by Thorsteinsson and Tillberg (1987), and Thorsteinsson et al. (1987) working with the classic clone of Le. gibba clone G3. The stress response of the plants was very similar towards both treatments. It was shown to be in two phases where, the first phase accumulated monosaccharides like fructose and glucose and the second one accumulated starch. On the whole, the growth of the fronds and their respiration rate were lowered and consequently also the usage of the products of photosynthesis for metabolism and growth. Consequently, starch accumulation in chloroplasts is unavoidable under these conditions. Interestingly, Thorsteinsson and Eliasson (1990) could not find any hint for the involvement of ABA under these nutrient-limiting conditions.

With the advent of the genomics era, transcriptomics of the plants (La. punctata) exposed to nutrient starvation could be used for investigations. Tao et al. (2013) and Li et al. (2021) revealed the upregulation of AGP (key enzyme of starch synthesis), and the contrary for the genes encoding starch degrading enzymes. However, as most of the ß-amylase enzyme is located in the cytoplasm without access to starch in the chloroplasts, the relevance of these results concerning starch-degrading enzymes is not clear. Under similar conditions, also, genes encoding the large and small subunits of AGP and that of the SSS were studied using proteomics and real time-PCR tools (Huang et al. 2014; Zhao et al. 2015). Under limitation of nitrate, it was shown that there is a hike in the activity of glutamine synthase which helps in reuse of the endogenous nitrogen in order to support plant growth as well as increase in starch content (Guo et al. 2020). However, it remains to be understood of how the transduction of the nutrient limitation signal leads to increased expression of starch-synthesizing enzymes and, consequently, in enhanced starch synthesis and accumulation.

Salt stress on starch accumulation

The impact of salt stress on duckweeds has been studied in several species and clones (Sree et al. 2015a; Ma et al. 2018). Apart from hindering the growth of the plants, salinity stress has induced accumulation of starch in duckweeds in a dose and time dependent manner. The exposure of duckweeds to saline conditions induced oxidative stress (Panda and Upadhya 2003; Cheng 2011; Cheng et al. 2013; Sikorski et al. 2013) and activated the antioxidant pathway to protect the plants from oxidative damage (Radic and Pevalek-Kozlina 2010; Chang et al. 2012; de Morais et al. 2019). The salinity treatment of W. arrhiza plants showed also an inhibition of photosynthesis with impaired electron transport and inactivation of PSII reaction centre (Wang et al. 2011). It is pertinent to mention here that the effects and their intensity very much depended on the species and clone and salinity dosage under study.

Interestingly, salinity stress on duckweeds did not have any impact on the uptake of nitrate from the medium. However, ammonium ion uptake was affected but only at salinity higher than 10 ppt (Nesan et al. 2020; there must be a mistake as this concentration is so low that any effect on plants could not be expected). At the initial time points of treatment within a day, a disturbance in the ionic homeostasis was observed with an increase in the Na⁺ content by 16 folds and a decrease in the K⁺ and Ca²⁺ content (Fu et al. 2019). Of interest is the increase of ABA levels by 150% with the treatment of duckweeds with 85 mM NaCl (Huber and Sankhla 1979) as it is known that ABA induces, very effectively, starch accumulation in duckweeds.

In the study performed by Fu et al. (2019) using S. polyrhiza 7498, it was reported that in the first few hours of treatment until 12 h, the starch content decreased and this correlated positively with the activity of AGP, the initial and rate-limiting enzyme of starch synthesis, which is involved in starch biosynthesis. A set of transcriptomic data with differentially expressing gene patterns, related to the time points within a day of treatment have also been presented. However, care must be taken to interpret these data, considering the day-length and possible interference by circadian effects on starch degradation (Lu et al. 2005). This circadian effect has been demonstrated by Ma et al. (2018) whilst screening a large number of clones belonging to Le. aequinoctialis and S. polyrhiza (together 20 clones) for starch accumulation. These authors could not demonstrate enhanced accumulation of starch under the influence of salinity on Le. aequinoctialis 6000 and S. polyrhiza 7498. Lack of accumulation of starch in four clones of S. polyrhiza under the influence of salinity was also detected by Sree et al. (2015a) although most of the other investigated species and clones accumulated starch within 7 days of treatment, e.g. Spirodela intermedia, La. punctata, Le. aequinoctialis and Le. minor. The highest starch content was detected in Le. minor 9441 after 14 days of application of 150 mM NaCl, 52% starch per DW.

Because of the growth inhibiting effect of starch inducing factors (as in case of salinity), Xu et al. (2011) using the method of nutrient limitation suggested a two-step procedure. Step 1 includes the cultivation of duckweed under optimal growth conditions to produce biomass. In step 2, the plants were transferred onto growth-limiting conditions to induce starch accumulation and to obtain finally large amount of duckweed biomass having a high content of starch. However, application of e.g. 50 mM NaCl does not inhibit growth much, in many species as demonstrated quantitatively by effective doses at 10, 20 and 50% inhibition. Nevertheless, high amounts of starch were accumulated under these conditions (Sree et al., 2015a). This holds true e.g. for S. intermedia 9394, Le. minor 9441 and La. punctata 0049. As a consequence, in case of salinity, it seems possible that duckweed cultivation (to obtain biomass) and induction of starch accumulation could be carried out in the same medium containing 50–100 mM NaCl without changing the medium. This would simplify the biotechnological application of salinity to induce starch accumulation in duckweeds.

Use of starch for production of bio-fuels

This part of the review focuses on the production of bio-alcohols using duckweed biomass. Methods for biofuel production from several floating aquatic plants by transesterification, pyrolysis, hydrolysis and torrefraction have been reviewed (Ma and Hanna 1999; Arefin et al. 2021; Djandja et al. 2021).

The group of Stomp and Cheng demonstrated not only the accumulation of starch in duckweed (e.g. S. polyrhiza) by extended cultivation in wastewater, but also reported on successful experiments to use the accumulated starch for the production of ethanol (Cheng and Stomp 2009; Xu et al. 2011; Cui and Cheng 2015). They commenced a two-step procedure by degrading starch to sugars (mainly glucose) enzymatically with α-amylase, pullulanase and amyloglucosidase (review in Cui and Cheng 2015). Since the publication of this pioneer work, several improvements have been introduced. Zhao et al. (2012) introduced cellulose enzymes to improve the yield of fermentable sugars obtained. They demonstrated that this process is very effective, but the enzymes they used are expensive. Treatment with pectinase also improved the yield (Chen et al. 2012). Being especially interested in the composition of the cell walls of five species of duckweed representative of all five genera, Pagliuso et al. (2020) treated the plants with the enzyme cocktail Cellic Ctec2 (Novazymes), with which only low recalcitrance to hydrolysis was observed. They assumed that this was due to the low contents of lignin and cellulose present in all duckweed species.

Acid hydrolysis is a viable alternative to enzymatic degradation to release sugar from starch (Cui and Cheng 2015). Rana et al. (2021) treated starch-rich S. polyrhiza for 1 h at 121 °C with 0.1% sulphuric acid and obtained 99.4% conversion of starch to sugar, mainly glucose. Su et al. (2014) compared acid hydrolysis and enzymatic degradation of La. punctata starch and found a higher sugar yield after acid hydrolysis.

The second step in the conversion of duckweed starch to biofuels, the fermentation of sugars to alcohols, is usually carried out with Saccharomyces cerevisiae, and ethanol is the main product (Xu et al. 2011; Cui and Cheng 2015). Higher alcohols were obtained by replacing this yeast with Clostridium acetobutylicum. Li et al. (2012) obtained a solvent phase containing about 68% butanol by treating La. punctata with C. acetobutylicum. La. punctata was also treated with a mutant of C. acetobutylicum or a bioengineered strain of Escherichia coli (Su et al. 2014). The main products obtained were iso-pentanol and butanol. Such higher alcohols are interesting as biofuels because of their higher energy content and lower degree of water contamination. Higher total yield was obtained by running starch saccharification and sugar fermentation simultaneously with La. punctata biomass (Souto et al. 2019).

There has been a recent tendency to improve the total energy output from starch-rich biomass by combining several biomass-processing technologies. Calicioglu et al. (2019) employed traditional fermentation followed by acidogenic digestion and methanogenic digestion. Combinations of hydrothermal treatment, anaerobic digestion and ethanol fermentation in different sequences were investigated recently by Kaur et al. (2019), and these authors found that hydrothermal treatment, followed by anaerobic digestion and completed by ethanol fermentation resulted in the highest energy output. An integrated approach in the sense of circular economy was also recently presented by Calicioglu et al. (2021) connecting wastewater purification by duckweeds and, transformation of starch and other components in a biorefinery to produce bioethanol as the main product beside fertilizer and biomethane.

Alternatively to the degradation of starch and fermentation of the released carbohydrates, duckweed biomass can be subjected to thermochemical conversion to produced bio-oil. Two different procedures are available, pyrolysis at high temperatures or hydrothermal liquefaction (Djandja et al. 2021). Compared to hydrothermal liquefaction, pyrolysis provides more aromatic hydrocarbons and less ketones resulting moreover in a bio-oil with lower N content, as reviewed recently by Djandja et al. (2021). Chen et al. (2020a, b) optimized the reaction conditions concerning temperature and duration of the treatment during hydrothermal liquefaction and reported that hydrothermal liquefaction at 370 °C for 45 min, a yield of nearly 41% was obtained. The heating value of the bio-oil was similar to that of petroleum.

Conclusions

The very high growth rates of several species and clones of duckweeds indicate that the chloroplastic starch that can accumulate in the fronds of these macrophytes can be of biotechnological relevance for the production of bio-alcohols via starch degradation and fermentation. However, the contents of the accumulated starch can be dramatically enhanced by appropriate manipulation of the cultivation conditions. It is advisable to implement the insights gained from investigations into the influence of ABA, uniconazole, heavy metals, nutrient deficiency and salt on the starch content of duckweed tissues on a large scale. Only then can the great potential of duckweeds as energy plants will be truly fulfilled.

Authors' contributions

All authors have designed the plan of the article, evaluated the literature and have written and proof-read the manuscript.

Funding

None.

Availability of data and materials

All data were given within the manuscript.

Code availability

Not applicable.

Declarations

Footnotes

References