3.2. Scientometric Analysis
A scientometric analysis was performed to analyze the authors’ impact. Out of a total of 482 authors having published documents in the field under study, 23 authors had published at least five documents, which were cited at least five times (
Table 2).
The average publication years of the published papers seem quite old (
Table 2). This might represent diminishing research; however, they are shown in relation to the citations. It also might have been caused by changing trends during the last years and a greater focus on physiology or genetics than practical applications. There is still a place for improvement of the plants related to applications such as biofuel production. Another reason is that researchers might be in the process of scaling up/or have already completed implementation research (industrial scale), and the results may not be available before they are patented.
The visualization of the authors’ co-citations is presented in
Figure 8. There are two clusters, which means there are two sets of closely related nodes that the VOSviewer software assigns to a network. Authors in one cluster are closely related and visualized on the map (
Figure 8). The node size indicates the number of citations obtained by an author. The larger the node, the more citations the author has received.
A scientometric analysis was also performed to analyze the published documents’ impact (
Table 3). Out of 141 documents published in the research area under study, 87 were cited at least 10 times, and 20 were cited at least 46 times.
Table 3 lists highly cited and the most influential articles based on the normalized number of citations.
The top three most cited documents were Xiong J.-Q. (2018), Cui W. (2015), and Muradov N. (2010) published in
Trends in Biotechnology,
Plant Biology, and
Bioresource Technology, respectively. The high number of citations might be related to the fact that each article is of the review type, not the research type. Another reason might be that the sources in which they were published are interdisciplinary. However, some of the most cited documents (Xiong J.-Q. (2018) and Chouhan A.P.S. (2013)) are not related to others (0 links). Among 87 documents, 82 are in the network and constitute 9 clusters (
Figure 9).
Out of a total of 78 sources, 11 sources published a minimum of 3 documents that were cited a minimum of 3 times.
Table 4 lists the most popular and highly cited sources. The top two are
Bioresource Technology and
Biotechnology for Biofuels. The next most popular and highly cited sources are
Biomass and Bioenergy and
Industrial Crops and Products. Those sources with six published papers each are in the third position. However, the number of total citations placed
Plant Biology with 154 citations in the third place among highly cited sources.
In
Figure 10, two clusters are presented based on co-citation visualization of the sources where analyzed documents were published in the studied research field.
With regard to analyzing organizations (337 in total), eight were in the network and had published a minimum of three documents cited at least three times. The
Chinese Academy of Sciences (Beijing, China) was the most productive. This organization was also the most cited in the analyzed sample. However, the average year of the published documents affiliated with this organization is 2017.55, which means that currently, researchers from this university might not be highly active in the study field (
Table 5.)
A visualization of the most influential organizations is presented in
Figure 11.
Out of thirty countries/regions involved in the research area under study, assuming that at least three documents were published, and each document was cited at least three times, nine meet the threshold (
Table 6). China was the most productive and cited 63 and 1572, respectively, which means that China is the most influential country in this field of study. However, the average year of publication is quite old (2014). The newest documents published (average year 2020.14) are affiliated with Brazil.
The visualization of influencing countries is presented in
Figure 12.
The full counting method in VOSwiever was used to create the network and visualization map of the most popular keywords. The minimum occurrence in each term in the analyzed documents was five. The terms with the same meaning, for example, ” bio-energy” and “bioenergy”, as well as words in singular or plural (“biofuel” and “biofuels”) were unified and replaced. Moreover, words without a connection, such as “article” and “priority journal”, were removed from the analyzed database. Out of a total of 1795 keywords, 114 terms were in relation to one another. As a result, visualization of the terms’ occurrence with six clusters was obtained (
Figure 13).
The first cluster (red) refers to hydrothermal liquefaction, pyrolysis, and bio-oil production. The second one (green) reflects the researchers’ focus on growth development using genetics, and the third one (blue) refers to anaerobic digestion and biogas, wastewater treatment, bioremediation, and phytoremediation. The fourth cluster (yellow) shows an interest of scientists in examining duckweed as an energy crop cultivation in the context of starch accumulation and production. The fifth cluster (purple) reflects studies on ethanol, fermentation, enzyme activity, saccharification, hydrolysis, and carbohydrate metabolism. The last cluster (turquoise) focuses on controlled studies on the toxicity of pollutants and their effect on plant growth.
Figure 14 presents a network map with the trend topics according to the keywords used in analyzed documents. The most recent keywords used in publications are indicated in yellow, and the oldest are indicated in purple. More recently published studies focused on wastewater treatment, anaerobic digestion, biogas production, feedstock, and phytoremediation. The appearance frequency of keywords is represented by circle size, and their correlation is represented by the distance between two circles.
3.3. Content Analysis
In-depth content analysis defined five research areas related to biofuels and duckweed:
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Starch accumulation and duckweed growth development.
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Development of the pretreatment techniques (e.g., enzymatic and acid hydrolysis).
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Ethanol fermentation, enzyme activity, saccharification, and carbohydrate metabolism.
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Hydrothermal liquefaction of duckweed biomass and bio-oil production.
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Anaerobic digestion and biogas production of biomass used for wastewater treatment, bioremediation, and phytoremediation.
Duckweed is the smallest and fastest-growing aquatic plant on earth, which can double its biomass in 16 h to 2 days and accumulate starch. Under optimal growth conditions, the dry weight of starch content can reach 75% [
68]. However, increased starch production in plants can be caused by stress related to nutrient deficiency; for example, the
Spirodela plants under nutrient stress enhanced their starch content from 21 to 80% [
71]. Tao et al. [
61], after a comprehensive gene expression profiling of
Landoltia punctata, exposed a bioengineered strain to nutrient deficiency. They suggested that the final starch accumulation was caused by the continuous intake of carbon, hydrogen, oxygen, and light combined with the suppression of many metabolic pathways and redirected metabolic flux. They revealed a decrease in the expression of key lignin biosynthesizing enzymes and an increase in the expression of transcripts involved with the synthesis of flavonoids [
61]. The light effect on
L. aequinoctialis 6000 biomass production was studied by Yin et al. [
59]. High light induction was responsible for starch accumulation in duckweed. They suggested that from an economical perspective, 110 μmol/m
2/s is the best light condition to obtain a high content of starch.
Starch accumulation by
Landoltia punctata might also be caused by heavy metals such as Co
2+ and Ni
2+. With the increase in the concentration of heavy metals, the activity of starch biosynthesis enzymes rises, and its accumulation grows in a very short time [
72]. The ability of duckweed to produce high biomass with a high starch content and a low lignin content renders it a raw material with high energy potential [
61].
Another factor that caused the rapid accumulation of high levels of starch in
Landoltia punctata is the application of plant growth retardant (uniconazole) [
73]. Starch accumulation resulted from the regulated expression of enzymes in the relevant pathways [
74].
Xu et al. [
75] studied the potential of different ecotypes in the turion of
S. polyrhiza strains and starch production. They obtained the highest starch productivity of 2.90 g/m
2/d. The authors demonstrated that after the two-step enzymatic hydrolysis, turion could be efficiently converted into ethanol (50.5 g/L) with a theoretical conversion rate of 91.67%. Moreover, they obtained an ethanol yield based on the initial turion biomass and starch at 0.34 g/g and 0.468 g/g, respectively. It is worth noting that a turion cultivation biomass yield of 13.8 t/hectare can be achieved. This yield can give an annual ethanol yield of 4.69 t/hectare, which is higher than the potential bioethanol yield of wheat and corn.
Some duckweed starch consists of 35.7% amylose and 64.3% amylopectin [
76], as well as the cell wall of cellulose (43.7%), pectin (20%), hemicellulose (3.5%), and lignin (about 3% for
Lemna minor) [
70]. This means biomass must be pretreated into simple sugars to be used in fermentation processes. For example, Su et al. [
77] tested five different methods of the enzymatic hydrolysis of
Landoltia punctata biomass for biobutanol production via fermentation. They tested different enzyme amounts and mixes, additives, temperatures and lengths of hydrolysis, and process pH values.
Duckweed biomass might be hydrolyzed using acidic treatment [
31,
78]. For example, according to Rana et al. [
31] very efficient hydrolysis of
Spirodela polyrhiza biomass by 0.1% sulfuric acid showed a conversion yield of starch to glucose of 99.4%. Hydrolyzed glucose was fermented with
Saccharomyces cerevisiae QG1 MK788210, and after 72 h fermentation under optimized conditions, authors obtained the highest amount of ethanol (1.21 g/L) from 2.2 g/L of glucose with 100% theoretical ethanol yield [
31]. Acid hydrolysis (1% H
2SO
4) was used to pretreat duckweed biomass for biohydrogen production through dark fermentation and simultaneously using the fermentative waste to produce microalgal lipids [
78].
Moreover, in the study performed by Rana et al. [
71], plant biomass of
Landoltia punctata with a high starch content was pretreated with diluted acid for conversion of starch to glucose and was next processed via fermentation with an indigenously isolated and optimized yeast strain. The process resulted in 99.8% of the theoretical ethanol yield. Interestingly, the authors proposed a closed loop and digested fermentation vinasse with a yield of 0.88 NL/g VS of biogas in anaerobic conditions. Analyses carried out on
Spirodela showed that the most energy is generated in producing biomethane and bioethanol, and in terms of energy efficiency, duckweed has a greater potential for biogas production. Pretreatment methods in the form of lyophilization and steam explosion were tested [
71].
Various methods for biomass pretreatment, such as the freeze-drying method and steam explosion, were also tested. Obtained hydrolysates were used to study the effect of pretreatment on the process of simultaneous saccharification using two commercial enzyme mixtures (Celluclast and CTec2) supplemented with additional β-glucosidase and fermentation carried out using the
Saccharomyces cerevisiae NCYC 2826 [
79]. Similar results of approximately 78% were obtained after 24 h incubation. However, doubling the concentration of enzymes did not provide any improvement to the yield. No β-glucosidase supplementation resulted in a decrease in yields of approximately 60%.
Hydrolysate from plants belonging to the
Lemnaceae family was the subject of the study to serve as a medium not only for ethanol but also for higher alcohol fermentation, for example,
Landoltia punctata hydrolysate. However, Su et al. [
80] used a bioengineered strain of
Corynebacterium crenatum with several inserted
Saccharomyces cerevisiae genes and a gene originating from
Lactococcus lactis cremoris via electroporation. Various modified bioengineered strains were able to produce higher alcohols. The yield of higher alcohols from the duckweed is extremely low (especially C5 alcohols such as 2-methyl-1-butanol and 3-methyl-1-butanol). According to the authors, further research will address the development of an optimal host for cell proliferation to best match the fermentation substrate of the duckweed and obtain the highest possible amounts of alcohols [
80].
Chen et al. [
81] indicated that the duckweed biomass (
Spirodela polyrhiza,
Lemna minor,
Landoltia punctata) from the sewage treatment plant could not only effectively remove nitrogen and phosphorus from water but also effectively produce starch, which renders it as suitable feedstock for the production of biofuel [
81].
Secondary effluents of municipal and swine wastewater, as well as effluent from anaerobic digestion diluted with tap water, were used by Toyama et al. [
67] for the grown biomass of
Spirodela polyrhiza,
Lemna minor,
Lemna gibba, and
Landoltia punctata to determine their starch production capabilities, caloric values for bioethanol, and biogas production. From one gram of dry biomass of
S. polyrhiza and
L. punctata cited, a range from 0.165 to 0.191 g of ethanol was achieved, and these results were higher than for
L. minor and
L. gibba. In the case of biogas production, potential varies between the biomasses of different species of
Lemnaceae.
To improve bioethanol yield, Ma et al. [
35] suggested that the selection of duckweed strains with high starch-producing ability is required. They tested 20 duckweed geographically isolated strains for biomass production, starch content, and starch production, and as a result, it was found that the best strain was
Lemna aequinoctialis 6000, with a biomass production of 15.38 ± 1.47 g/m
2, a starch content of 28.68 ± 1.10%, and starch production of 4.39 ± 0.25 g/m
2 [
35]. Biomass (containing 34% of starch) from the aforementioned strain grown on sewage was used by Yu et al. [
82] in a one-step enzymatic saccharification process with an over 94% recovery yield. They obtained 0.44 g ethanol per g of glucose with a common yeast strain (Angel yeast).
Gusain and Suthar [
83] grew locally found strains of
Lemna gibba,
Lemna minor,
Pistia stratiotes, and
Eichhornia sp. in 500 L containers in tap water with wastewater and a piece of cow manure as a nutrient source. The obtained biomass was tested for enzymatic saccharification of powdered dry biomass for bioethanol fermentation efficiency. All tested duckweed species have a similar yield of ethanol per unit of biomass from 0.189 g/g (
Eichhornia sp.) to 0.218 g/g for
L. minor [
83].
When four species of duckweeds (
Landoltia punctata,
Lemna aequinoctialis,
Spirodela polyrrhiza, and
Wolffia arrhiza) after enzymatic pretreatment (a-amylase and amyloglucosidase (NovozymesTM)) were used in the test of the fermentation process with yeast S.
cerevisiae, the final ethanol concentration was from 0.17 to 0.19 g ethanol/g dry biomass [
76].
The ethanol yields for untreated, freeze-dried, and steam-exploded were 31.4%, 61.3%, and 78.5%, respectively, of the calculated theoretical maximum. Although the ethanol yield from the exploded biomass was high, the final concentration in the medium was 0.25% (
v/
v), whereas concentrations generally considered viable for distillation must exceed 4%. Moreover, increased stirring and addition of different amounts of yeast that was preconditioned for natural inhibitors resulted in an increase in yields of up to nearly 70% of the theoretical maximum (13.5% g/g dry matter) [
79].
Pretreated Lemnaceae was fermented to butanol and isopentanol by
Saccharomyces cerevisiae strains,
Clostridium acetobutylicum, and bioengineered
Escherichia coli [
18]. The yields obtained for ethanol and isopentanol from acid hydrolysate were 15 times higher than what could be obtained through the fermentation of the yeast mutant. The authors confirmed that it is possible to obtain butanol, isopentanol, and pentanol from the acid-hydrolyzed biomass via fermentation by the bioengineered strains of
E. coli. [
18].
Hydrothermal pretreatment was the subject of the study by [
69,
70,
71,
72]. Kaur et al. [
84] combined hydrothermal pretreatment and anaerobic digestion to improve the enzymatic digestibility of biomass by facilitating the maximal removal of hemicellulose (68.5–73.5%). As a result, glucose production was 36.5–44.2 g/biomass, ethanol yield 0.167–0.231 g/g biomass, and methane yield 32.9–52.5 m
3/ton. The authors showed that the integration of both processes, i.e., anaerobic fermentation and ethanol fermentation in a biorefinery, will allow for the achievement of higher energy efficiency [
84].
A combination of freeze milling and microwave hydrothermal pretreatment (130 °C to 210 °C, 10–40 min) was applied for the preprocessing of
Landoltia punctata biomass, and its effects on the process of bioethanol production were studied by Souto et al. [
85]. As a result, insoluble materials in the biomass were significantly reduced. After the higher severity treatment (210 °C for 40 min), biomass contained 48.8% of insoluble material, whereas biomass pretreated in lower temperatures (130 °C for 10 min) contained about 69.2%. A decrease in starch and an increase in monosaccharide concentration for the treated biomass were also observed. It is also reported that approximately 67% of hemicellulose was solubilized at the highest severity. Pretreated biomass was used in a simultaneous saccharification and fermentation process with CellicTM CTec2 cellulase and the
Saccharomyces cerevisiae NCYC 2826 strain, and the maximal yields of ethanol were achieved at 88.81 wt.% for biomass pretreated at 200 °C for 10 min [
85].
Gaur et al. [
69] pointed out that the thermal pretreatment caused a positive effect on chemical dynamics and CH
4 production in a digester. They demonstrated that
Lemna minor is a promising feedstock for biomethanation if mixed in the appropriate proportion (50–60%) in sludge. The maximum CH
4 yield was 468 mL CH
4/g VS in DW. Thermally treated setups showed higher CH
4 than non-treated setups.
Researchers also studied the hydrothermal liquefaction of duckweed [
86,
87]. For example, Chen et al. [
86] explored potential catalysts for upgrading duckweed (
Lemna minor) biocrude in subcritical water. The most active catalyst was Ru/C, which obtained liquid fuel with properties similar to those of hydrocarbon fuels derived from fossil fuel resources. The upgraded oil was characterized by low viscosity and high energy density, making it suitable for the co-feedstock in a conventional refinery to produce transportation fuels.
Hydrothermal liquefaction at temperatures of (250 °C–370 °C) and times of (15–60 min) was tested by Chen et al. [
87] for bio-oil production from duckweed biomass after phytoremediation. They obtained the highest bio-oil yield of 35.6 wt.% at 370 °C after 45 min pretreatment. Moreover, the higher heating value of bio-oil was 40.85 MJ/kg, and the H/C ratio (1.72–1.98) was similar to that of petroleum (1.84).
Lemna minor was assessed for its potential as a feedstock for gaseous fuel production (bio-hythane-hydrogen and methane) in an integrated strategy. Kaur, Srikanth, et al. [
88] applied three approaches: acidogenic fermentation, electrohydrogenesis, and methanogenesis, which were evaluated in a single stage and in different combinations of two and three stages to tap the maximum feasible energy. The single-stage processes were insufficient in substrate degradation and its energy conversion. The authors decided that to increase energy conversion efficiency, bioprocesses should be integrated with acidogenic fermentation as an initial stage. The acidogenesis causes a significantly increased content of VFA and H
2, which can be an additional source of biofuels [
88].
When
L. minor was used in the feed mixture, there was a clear improvement in the gas production rate (40%) and methane-specific production (41%) compared to mono-substrate digestion [
42]. The proposed co-digestion is in line with the most recent trends regarding resources and waste valorization, which aim to promote a circular economy, recovering energy, water, and nutrients from swine wastewater [
42].
Anaerobic co-digestion processes using
Lemna minor biomass with manure and food waste were also studied by Chusov et al. [
89] to determine biogas potential (biogas volume, methane content). The obtained results confirm the possibility of using this type of waste for biogas/biomethane production.
Aquatic plants, including
Azolla filiculoides and
Landoltia punctate, are high in carbohydrates and can also be used for the phytoremediation of industrial wastewater. Their biomass has been used as an alternative carbon source for biodiesel production. The aforementioned biomass might also serve as a raw material for bio-hydrogen fermentation using
Enterobacter cloacae [
90].
The dried biomass of
Lemna gibba, which was cultivated on urban wastewater, showed a high content of total sugar (38.0%), starch (24.5%), and lipid (9.3%). The extracted lipid showed high contents of C16:0-palmitic acid (37.68%), C18:2-linoleic acid (18.11%), and C18:3-linolenic acid (33.76%). The heating value ranged between 15.07 and 18.58 MJ/Kg, and it was in higher ranges as per standards [
91].
Duckweed biomass has also been considered feedstock for producing advanced biofuel precursors. Calicioglu et al. [
92] studied the effect of operating conditions (i.e., mesophilic (35 °C) or thermophilic (55 °C) conditions; an acidic (5.3) or basic (9.2) pH) on the yield and composition of the end products from wastewater-derived duckweed during acidogenic digestion. Operating conditions significantly affect the end product resulting from the acidogenic digestion of duckweed.
There have also been studies on dark fermentation for biohydrogen production using an acid hydrolysate of duckweed biomass. Mu et al. [
78] achieved a maximum hydrogen production of 169.30 mL/g DW at 35 °C and an initial pH of 7.0.
The production of biofuels highly depends on costs. As Yu et al. [
82] pointed out, high medium costs and ingredients might render cultivation not economical. Furthermore, the cost of the biorefining process might increase due to the high moisture content of fresh duckweed biomass [
70], which results in additional energy consumption for drying [
93]. During biorefinery operations, it is important to maintain an uninterrupted supply chain. Duckweed is considered an inexpensive, sustainable source of plant biomass for biofuels [
18].
Furthermore, duckweed is cheaper than straws of cultivated plants [
93]. However, there are additional costs, such as transport costs, the risk of biomass spoilage, and energy for drying. Moreover, the capital and operating costs of a biorefinery depend on the area (rural, urban), the price and availability of land for duckweed cultivation, and differences in the concentrations of inflowing sewage [
93]. Another economic barrier hindering the commercialization of lignocellulose-based biorefinery is extensive water consumption during the cultivation of feedstock and biofuel processing [
84].
Patel and Bhatt [
94] provided a circular economic model for the utilization of duckweed for sustainable feedstock for biofuel and a variety of natural products described, such as pigments, lipids, and nanocatalysts in an integrated manner from fresh biomass
S. polyrhiza, with a small amount of generated waste. One ton of
S. polyrhiza biomass gave 0.8–1.2 kg of R-phycoerythrin, 0.7–0.9 kg of R-phycocyanin, 2.7–4.3 kg of lipids, 5.3–6.1 kg of zerovalent Iron, 79.7–80.4 kg of starch. The starch was fermented to ethanol with a yield of 38.8–40.8 L. The waste generated in each step produced 2.23 L biogas equivalent and 8.51 GJ energy. The residues were reduced by 79–85% in chemicals and energy usage during starch extraction.