Valorization of Spirodela polyrrhiza biomass for the production of biofuels for distributed energy

Considering the main objectives of a circular economy, Lemnaceae plants have great potential for different types of techniques to valorize their biomass for use in biofuel production. For this reason, scientific interest in this group of plants has increased in recent years. The aim of this study was to evaluate the effects of salt stress on the growth and development of S. polyrrhiza and the valorization of biomass for biofuel and energy production in a circular economy. Plants were grown in a variety of culture media, including standard 'Z' medium, tap water, 1% digestate from a biogas plant in Piaszczyna (54° 01′ 21″ N, 17° 10′ 19″ E), Poland) and supplemented with different concentrations of NaCl (from 25 to 100 mM). Plants were cultured under phytotron conditions at 24 °C. After 10 days of culture, plant growth, fresh and dry biomass, as well as physio-chemical parameters such as chlorophyll content index, gas exchange parameters (net photosynthesis, transpiration, stomatal conductance and intercellular CO2 concentration), chlorophyll fluorescence measurements were analyzed. After 10 days of the experiment, the percentage starch content of Spirodela shoot segments was determined. S. polyrrhiza was shown to have a high starch storage capacity under certain unfavorable growth conditions, such as salt stress and nutrient deficiency. In the W2 (50 mM NaCl) series, compared to the control (Control2), starch levels were 76% higher in shoots and 30% lower in roots. The analysis of the individual growth and development parameters of S. polyrrhiza plants in the experiment carried out indicates new possibilities for the use of this group of plants in biofuel and bioethanol production.

The increasing demand for alternative energy sources has caused a significant development of technologies for obtaining renewable fuels derived from biological raw materials.Reduction of greenhouse gas emissions, biodegradability, diversification in fuel sectors, development of the agricultural products market and sustainable development are the main reasons for increasing the expenditure on obtaining renewable energy from liquid fuels such as bioethanol and biodiesel.On the other hand, bio-waste treatment processes, including wastewater treatment plants, biogas plants and composting, should be adopted to prevent bio-waste from going to landfills, protect the environment and comply with European environmental policy 1,2 , and, if possible, convert it into biofuels and renewable energy to reduce climate change 3 .Using different pre-treatment techniques we can valorize specific biomass for specific purposes-for example the torrefaction process can significantly increase the amount of carbon in solid biofuel 4 .
Starch next to cellulose is one of the most common biopolymers on earth.It is not only one of the main elements of the everyday carbohydrate diet of every person, but above all it is a very important substrate used for food, paper, pharmaceutical, and textiles.In recent years it is used in the energy industry for the production of fuels such as bioethanol 5 .Starch is an insoluble polysaccharide, a polymer of glucose residues synthesized in chloroplasts of higher plants, acting as a storage of energy generated during photosynthesis 6 .This sugar is produced during the day in plant organs characterized by high photosynthesis intensity, and then used at night to support continuous metabolism, therefore starch is considered as one of the basic factors regulating plant growth 7 .In

Materials and methods
Plant collection, culture, exposure conditions and experiment variants.The experiment was conducted for 10 days under laboratory conditions on the model water plant S. polyrrhiza, derived from the collection of in vitro cultures of the Department of Plant Ecophysiology of the Faculty of Bioscience of the University of Lodz, Poland.Plant samples for testing were collected and evaluated according to methodologies developed on the basis of national standards and previous research, which are in force at University of Lodz in Poland and have been used in previous studies and international publication 19 .Test were conducted in accordance with OECD 221 Assess the toxicity of substances to freshwater aquatic plants of the genus Lemna (duckweed).
The plants were cultured in the presence of different concentrations of NaCl and in control variants.Morphological observation was carried out daily throughout the experiment.There were three control variants (I) standard "Z" liquid medium (pH 6.4), (II) tap water (pH 7.0) and (III) tap water supplemented with 1% postfermentation effluent from a biogas plant in Piaszczyna, Poland (pH 8.1).Medium "Z" is a standard medium for growing in vitro culture plants containing all necessary micro and macro elements.Plants were cultivated for 10 days in a phytotron room at 24 °C, under constant lighting with PHILIPS MASTER TL-D lamps with a power of 2 × 18W/840.The pH of the liquid medium "Z" was determined using a Seven Compact™ S210 pH meter.Spirodela sp. were grown in 250 mL Erlenmeyer flasks with 100 mL liquid medium.

Treatments and experimental design
The experimental medium was prepared on the basis of previous results from a macrophyte culture obtained at the Department of Plant Ecophysiology at the University of Lodz.The experiment was carried out according to the following experimental variants: Control series: • Control 1-100 mL standard "Z" medium   Equation (1) Formula for calculating the percentage of starch in samples according to the method of the manufacturer Starch Assay Kit from Sigma-Aldrich, product Code STA20.

Statistical analysis
Statistical analyses were performed with the Statistica 12 program.The data are expressed as mean ± standard deviation (SD) of 10 independent experiments.The normality of the data distribution was assessed by Kołmogorov-Smirnov test.The homogenicity of variances was determined using Levene'a test.Differences between samples were analyzed using one way parametric analysis of variance (ANOVA) with the post-hoc Dunnett's test p values ≤ 0.05 were considered significant.

Relative growth rate
Daily observation of morphological features, counting of new shoot segments, physical and chemical analyses performed on the last day indicated the varied sensitivity of S. polyrrhiza plants grown on the medium supplemented with various concentrations of NaCl and leachate after methane fermentation.RGR (Relative Growth Rate) is shown in Fig. 5I.When analyzing the kinetics of plant growth, all fronds that were visible, regardless of their size, were taken into account.Based on the number of fronds on the "zero" day (start day of the experiment) and the 10th last day, relative growth rate (RGR) was calculated according to the formula: x1-is the number of fronds on the zero day of the test, x2-is the number of fronds on the tenth day of the test, t-specifies the number of days of breeding.
Equation 2 Formula for calculating Relative Growth Rate (RGR).
The series run on "Z" medium supplemented with 75 mM NaCl (Z3), with plant growth 7.5% higher than the control (Control1) was the most effective.In the variant with 1% post-fermentation effluent, the number of macrophyte fronds was higher by 15% (O1-25 mM NaCl) and 7% (O2-50 mM NaCl) compared to the control series (Control2).The beneficial effect of NaCl was also observed in the series with tap water alone, where the plants grown on the medium supplemented with 25 mM NaCl (W1) and 50 mM (W2) showed 3-5% better growth compared to Control3.

Index of chlorophyll content
Similar results were obtained when analyzing the chlorophyll content index of Fig. 4I,II.The highest concentration of chlorophyll in Lemnaceae shoots (fronds) was obtained in the variant with "Z" standard medium supplemented with 75 mM NaCl (Z3).In the other variants the values were similar to the control samples.

Gas exchange parameters
On the tenth day of the experiment, several biochemical analyses were carried out, gas exchange parameters were determined, including the intensity of photosynthesis in the fronds of S. polyrrhiza plants.In the series of macrophytes grown on "Z" standard medium, the most favorable effect and value of net photosynthesis (Fig. 5I) with 100 mM NaCl.The value of net photosynthesis in the plants grown on medium with the addition of 1% post-fermentation effluent was characterized by a decrease that was inversely proportional to the concentration used, and so the intensity of the net photosynthesis process compared to the control (Control3) was successively lower in the O1 variant 25 mM NaCl by 3%, in O2 and O3 (50 mM and 75 mM) by 6.5%, and in the O4 series (100 mM NaCl) lower by 13%.The intensity of the transpiration process observed in all variants grown on "Z" medium (Fig. 5II) remained at a level similar to control (Control1) and ranged from 3 to 3.2 mmolH 2 O/m 2 s 1 .A different tendency was demonstrated by the series grown on the medium with 1% post-fermentation effluent with tap water.Transpiration was characterized by a greater intensity compared to the control (Control2): O1 and O4 variants (25 mM and 100 mM NaCl) higher by 3.5% and O2 and O3 variants (50 and 75 mM NaCl) higher by 10%.
The stomatal conductivity (Fig. 5III) in the variants with the "Z" standard medium was similar and ranged from 876 to 902 molH 2 O/m 2 s 1 .This parameter was different in the series carried out on tap water with the addition of NaCl, variant W1 and W2 (25 and 50 mM NaCl) remained at a similar level as control (Control2) while W3 and W4 (75 and 100 mM NaCl ) were higher than the controls by 7 and 6.5% respectively.In the series with 1% leachate, the values of stomatal conductivity were 5.5% higher O1 (25 mM) and 4.5% lower O2, O3 and O4 (50, 75, 100 mM NaCl compared to the control (Control3).
In contrast, the highest content of intercellular CO 2 was determined in the series with tap water supplemented with: 25 mM NaCl (W1), 50 mM NaCl (W2), 75 mM NaCl (W3).The values obtained were quite similar and ranged from: 671-721 compared to the control (691), with variant W1 having a higher value than control 2. It should also be noted that all experimental variants containing 'Z' medium had the lowest CO 2 content values compared to the series containing tap water and 1% leachate, indicating a more efficient photosynthetic process (Fig. 5lV).

Chlorophyll fluorescence and fresh biomass
Chlorophyll fluorescence (Fig. 6I) and fresh biomass (Fig. 6II) were also analyzed.Analysis of the fluorescence of the dark-adapted sample versus maximum fluorescence (maximum PSII photosystem efficiency (Fv/Fm) made it possible to determine the relationship between the structure and function of the photosynthetic apparatus and to estimate the vitality of the plants.Shoot segments were adapted in the dark to ensure that all the reaction centers in photosystem II were opened (oxidized) and ready to receive electrons before the measurement.The highest chlorophyll fluorescence was characteristic of the series of plants grown on "Z" standard medium.The Fv/Fm

Concentration of starch
The starch content (Fig. 7) in Lemnaceae plants was determined separately for shoots and root.Plants grown only on tap water and supplemented with NaCl (food stress-medium without nutrients and salt stress) were characterized by a high concentration of starch.In the W2 series (50 mM NaCl) compared to control (Control2) the starch level in the shoots was higher by 76% and in the roots lower by 30%.The starch contents in the other variants with tap water compared to the control (Control2) were as follows in the shoots: W1 (25 mM NaCl) higher by 14%; W3 (75 mM NaCl) higher by 3%; W4 (NaCl) higher by 10%; in the roots: W1 (25 mM NaCl) lower by 32%; W3 (75 mM NaCl) was lower by 56%; W4 (NaCl) higher by 7%.The macrophytes grown on the medium with 1% post-fermentation effluent supplemented with NaCl compared to the control (Control3) were characterized by varying levels starch content in the shoots compared to the control for the O1 series (25 mM NaCl) was 34% lower; for O2 (50 mM NaCl) 61% lower; for O3 (75 mM NaCl) 42% lower; for O4 (100 mM NaCl) 51% lower.Starch content in the roots for the O1 series (25 mM NaCl) was 37% lower for O2 (50 mM NaCl) 69% lower; for O3 (75 mM NaCl) 27% lower; for O4 (100 mM NaCl) 10% higher.
Spirodela plants growing on "Z" standard medium with the addition of various concentrations of NaCl showed high sensitivity to salt stress, which was reflected in the level of starch production and storage in shoot segments.Compared to control (Control 1), the starch level in the shoots was lower and was as follows: for the Z1 series (25 mM NaCl) it was 42% lower; for Z2 (50 mM NaCl) it was 29% lower; for Z3 (75 mM NaCl) it was 21% lower; for Z4 (100 mM NaCl) it was 34% lower, in the roots: for Z1 series (25 mM NaCl) it was 89% lower for Z2 (50 mM NaCl) it was at the same level; for Z3 (75 mM NaCl) it was 11% lower, for Z4 (100 mM NaCl) it was 61% higher.

Discussion
The presented results of our research relate to the result of the work of many authors, including Stelmach et al. ( 2021) and Szufa et al. ( 2019) who obtained interesting results in obtaining biomass from watercress.The authors of the above work obtained a biomass yield of 39.1-105.9t ha −1 year −1 of water lash, and this was achieved using leachate as a nutrient source, and the physiological parameters determined in the plants were significantly higher than potential energy plants 42,50 .Like other authors, we also showed in our study that S. polyrrhiza has a high starch storage capacity under salt stress conditions 42,43 2021) proved that adverse factors such as salt stress, sudden temperature drop, higher daily light intake (DLI) or nutrient deficiency, promoted starch production and accumulation.They proved that the starch content of watercress grown at 5 °C was 114% higher than that of watercress grown at 25 °C, and confirmed that photosynthetic efficiency depends on light availability (increase in DLI) and temperature.The results of their experiment indicated that starch production varied with temperature from 5 °C to 20 °C.The above authors, by changing the culture conditions from nutrient-poor medium to tap water, achieved an increase in starch content by 26.6% 51 .Similar results were obtained in our work using tap water as a medium supplemented with 50 mM NaCl.We obtained significantly higher starch compactness in shoot members compared to a series of plants grown on water alone.On the other hand, Toyama et al. (2018) studied four species of water cilia (S. polyrrhiza, Lemna minor, Lemna gibba and Landoltia punctata) and confirmed their ability to remove nitrogen from the environment 52 .They cultured these macrophytes for 4 days on municipal waste, animal husbandry waste and after anaerobic digestion.They proved that biomass production and nitrogen removal efficiency from all three types of liquid waste were most effective for S. polyrrhiza.Growth rates of all four species of water lash were higher on nitrogen-rich wastes, i.e. animal waste and anaerobic digestion wastes, than on municipal wastewater.It should also be noted that ethanol and methane production was higher from the biomass of S. polyrrhiza and L. punctata grown on each leachate compared to the other substrates 51,53 .
The ethanol production potential of some Lemnaceae species grown in ponds with leachate has been shown to be comparable to waste biomass (sugarcane), intercrops (alfalfa fiber) 47 , aquatic plants (water hyacinth and water lettuce) 54 and Lemna minor 25,52 , Wolffia globosa 51,55 or Wolffia arrhiza 56 , and microalgae such as Chlamydomonas reinhardtii 57 and Chlorella vulgaris 28,[57][58][59] .Our study confirmed that salt stress differentially affected the level of starch in the shoots and roots of S. polyrrhiza and was dependent on the concentration of NaCl.
In addition, macrophytes growing in polluted water bodies with high salinity levels can not only produce large amounts of biomass through starch storage, but can also reduce the level of salinity in the environment.Since Lemnaceae strains have been proven to have varying sensitivity to the amount of stored starch, strain optimization and ecotoxicological analyses of individual Lemnaceae strains are needed before they can be used on a large scale in the energy industry.
The results obtained in our study confirmed previous literature reports and indicated the relationship between starch accumulation in plants and the effect of salt stress on them.Similar findings were presented by Xu et al.  (2011), who analyzed the effects of 10, 20 and 30 mM NaCl on plant growth, as well as on starch content, which was 13.4 and 18.7% higher than in the control sample, respectively 16 .On the other hand, Sree et al. (2015), in a study of four strains of S. polyrrhiza, showed a slight increase in the starch content of the cells, but in this experiment the level of NaCl used was much higher at 450 mM 18 .Grajek et al. (2008) pointed out that access to low-cost starch and cellulosic feedstocks is crucial for bioethanol production.The development of innovative plant growth technologies for bioethanol production is a priority for the distilling and energy production industry 60 .Yin et al. (2015) analyzed biomass productivity in relation to bioethanol production and showed that it was not the quantity of biomass, but its quality, including plant starch content, light conditions and photoperiod, that was most important.It was shown that a 14-percent increase in grain yields due to fertilization with 120 kg Nxha −1 translated into increased ethanol production of 254.2 Liters per hectare 61 .S. polyrrhiza, on the other hand, could be a valuable feedstock for bioethanol production in the future due to its rapid biomass growth and high starch storage capacity 23,62 .However, the degree of starch www.nature.com/scientificreports/production and accumulation depends on the species.In a study conducted by Ma et al. (2018) in 2017, different strains of L. aequinoctialis and S. polyrrhiza isolated from different latitudes were selected to determine their potential for bioethanol production 22 .The most favourable results were obtained with L. aequinoctialis 6000 plants, where biomass production was 15.38 ± 1.47 gm −2 , starch content was 28.68 ± 1.10%, and starch production was 4.39 ± 0.25 gm −2 .This strain had the highest starch production after 8 h of exposure, and tap water proved to be the most favorable substrate; interestingly, salt stress in the form of NaCl did not induce starch accumulation in plant cells.Sree et al. (2016) using 16 varieties of macrophytes Lemnaceae indicated that salt stress had a positive effect reflected in increased starch accumulation in plants, however, some stressors (also NaCl) could inhibit the vegetative development of duckweed more effectively than a decrease in photosynthesis.Photosynthetic low carbohydrate production leads to low utilization by growth-related metabolism 17 .However, the varying range of plant responses to different concentrations of NaCl causing growth inhibition dependents on the species of plant and may indicate that macrophytes have alternative mechanisms for the use of excess energy.
Xu et al. (2011) used the two-step method to increase the starch content in S. polyrrhiza strain.First, the duckweed was grown under optimal conditions (nutrient-rich water) to produce a large amount of biomass, and then the plants were transferred to growth-limiting conditions (distilled water), resulting in the accumulation of starch.Sree et al. (2015) proposed an alternative one-step method of limiting the growth and stimulation of starch storage in water duckweed based on the use of salt stress in the range of 50 to 100 mM NaCl.Our results presented in this paper confirm these observations.In our experiment 50 mM NaCl most effectively stimulated starch accumulation in S. polyrrhiza.
In our laboratory experiments, we obtained 10% starch content (tap water plus a variant of 50 mM NaCl).We suggest that future experiments should take into account the different adaptive strategies of Lemnaceae to salt stress.The increased starch content observed in our experiment at lower NaCl levels, as in other studies, may be attributed to metabolic modification toward resistance or reduced toxicity of the stress factor.These processes may be related to the adaptive photosynthetic apparatus (mainly PSII), synthesis of various compounds, including glycerol, sugar, other osmoprotectants and specific proteins 14,52 .Increased NaCl in the medium also inhibits photosynthesis in Wolffia arrhiza, as evidenced by impaired electron transport and inactive photosystem II reaction centers 31 .
In our study, the decrease in the starch content in the roots was observed at the concentration of 100 mM NaCl.Salinity stress-induced inhibition of vegetative growth was reported for several duckweed species, including S. polyrrhiza, L. minor and L. gibba 17,18 .These observations may lead to the conclusion that the increase in salinity induces osmotic stress 26 and activates the antioxidative plant defense system 63,64 .Similar effects were observed during starch analysis in plant roots 53 .
NaCl increased starch accumulation is mainly due to osmotic stress caused by primary stress.Plant responses to osmotic stress are regulated by abscisic acid (ABA).Liu et al. (2018) proved that high biomass growth and starch accumulation in L. punctata plants were caused by abscisic acid 53 .ABA supplementation increased the percentage of starch from 2.29% to 46.18% after 14 days, with a total starch level 2.6-fold higher compared to the control group.They showed that endogenous ABA increased biomass efficiency production and promoted the accumulation of starch by duckweed, but also affected the level of other endogenous hormones, it increased zeatin riboside and indole-3-acetic acid, and decreased gibberellin.In addition, abscisic acid regulated the activity of enzymes involved in starch biosynthesis and duckweed catabolism.The activity of enzymes involved in starch biosynthesis increased, while the activity of enzymes catalyzing starch degradation decreased after using ABA.Liu et al. (2018) concluded that ABA might promote biomass and starch accumulation regulating endogenous hormone levels and the activity of key enzymes associated with starch metabolism 53 .
Rapid growth of high-quality duckweed biomass is considered a key step to its use for the production of biofuels.Under ideal conditions, water duckweed could double its biomass in 16-48 h.The rate of growth of water duckweed can reach 12.4 g/m 2 /day of dry matter, and its efficiency was documented at 55 tons/ha/dry weight 26 , so much higher compared to the energy plants used so far.
The experiments showed multidirectional productivity of aquatic plants Lemnaceae Fig. 8. High biomass content in the plants cultivated on the medium supplemented with postfermentation leachate indicated the possibility of using effluent from biogas plant.The use of leachate in the designed cost-efficient culture medium increases profitability and competitiveness of a biorefinery with regard to ecologic and environmental issues.Management of biogas plants wastes will allow to diminish ecological footprint of this type of installations and to significantly decrease bioethanol production costs.
All-year vegetation period, quick and high biomass yield, cost-efficient production and high starch content make water plant Lemnaceae an alternative to the plants now used for bioethanol production (corn, cereals, sugar beet).Production of energy plants poses many problems including limitation of the area for their growth, harmful effect of adverse weather conditions, high water consumption.The production of the water plants Lemnaceae with the use of biogas plant waste meets the requirements of sustainable technology of renewable energy resources and sustainable recycling economy 4,58,65 .
The high (up to 70%) starch content and low content of lignin and hemicellulose in Lemnaceae as compared to the other energy plants, from the technological point of view is much more effectively used for bioethanol production.They do not require preliminary heat treatment, which in standard procedures is time consuming and costly.Lemnaceae biomass is produced in bioreactors or in open water bodies.It can be used as a multiprotein supplement of animal feed, as a bioindicator to assess environmental pollution and in phytoremediation during processing of municipal and agricultural waste.
The obtained results indicated that Spirodela sp. in response to abiotic stress such as high salinity of the environment was able to increase starch production in comparison with other popular energy plants.The use of duckweed as a source of starch for the production of bioethanol allows to create a wide spectrum of new possibilities for this group of plants.High accumulation of starch with low lignin content increased ability to absorb nutrients such as nitrogen and phosphorus from leachate, increased absorption of CO 2 due to intensified photosynthesis make S. polyrrhiza, similarly as other water duckweeds, a promising raw material for the production of biofuels 66 .Our experiment confirms the effective use of aquatic plants in the production of biofuels, which creates opportunities for the development of innovative, alternative and cost-effective energy sources, and is confirmed by the 2023 review publication.

Conclusion
In recent years, scientific advances in the energy sector have fundamentally contributed to the development of several environmentally friendly technologies and processes, particularly in the production of biofuels including bioethanol.These solutions allow the production of biofuels from plant biomass and the development of clean green technologies.In recent years, there has been a growing interest in the production of bioethanol as a liquid fuel for powering motor vehicles.Ethanol can be produced from products containing monosaccharides as well as polysaccharides including starch and represent promising feedstocks for larger-scale bioethanol production.Technological advances are enabling improved production techniques and cost reductions that will favor the production of bioethanol from S. polyrrhiza plants as a fuel, especially in view of the benefits that its potential production entails in terms of less environmental contamination, agricultural activation and reduced oil import costs.
Control 2-100 mL of tap water • Control 3-99 mL of tap water + 1 mL of fermentation effluent from a biogas plant (1% leachate-the optimal concentration obtained on the basis of previous experiments.This leachate concentration does not require preliminarny plant adaptation ) Different variants of medium supplemented with NaCl: Figures 1, 2, 3.

Figure 2 .
Figure 2. Growth kinetics of Spirodela polyrrhiza plants (after 10 days) grown on different nutrient variants: Control with 2-100 mL of tap water (I); tap water supplemented with 25 mM NaCl (II), tap water supplemented with 50 mM NaCl (III), tap water supplemented with 75 mM NaCl (IV), tap water supplemented with 100 mM NaCl (V).

Figure 8 .
Figure 8. Circular economy concept for valorization of Spirodela polyrrhiza biomass to produce different products: solid and liquid biofuels.