Effects of waterweed compost derived from Lake Biwa on Komatsuna (Brassica rapa var. perviridis) growth

ABSTRACT In Lake Biwa, located in Shiga Prefecture, Japan, the overgrowth of waterweeds has become a significant environmental and human-life problem. Therefore, the waterweeds are systematically harvested and composted. In order to consider the effective use of waterweed compost for carbon neutrality and resource recycling, we clarified the characteristics of the waterweed compost by cultivation experiments with Komatsuna (Brassica rapa var. perviridis). When the waterweed compost (20%) was applied alone, the growth of plants was promoted about 1.7 times compared to a control containing the same amount of fertilizer components. However, the plant showed yellowing of leaves and a high C/N ratio, indicating obvious symptoms of nitrogen deficiency. The application of the waterweed compost (20%) with chemical fertilizer remarkably enhanced plant growth up to about 3.5 times without nitrogen deficiency compared to a control containing only chemical fertilizer. Interestingly, the coexistence of the waterweed compost and chemical fertilizer activated nitrification and diversified soil bacteria (Chao1 index, 213.0; Shannon index, 6.50) rather than the waterweed compost alone (Chao1 index, 41.3; Shannon index, 4.34). Our results indicate that the waterweed compost functions effectively as an organic fertilizer and a soil amendment, contributing to sustainable agriculture. GRAPHICAL ABSTRACT


Introduction
The overgrowth of waterweeds has become a significant global environmental problem (Hussner et al., 2017;Santos et al., 2011). The overgrowth of waterweeds has also significantly altered the natural environment and ecosystem in Lake Biwa, located in Shiga Prefecture, Japan. In addition, it has obstructed fisheries and ship navigation, causing a rotten stink of waterweeds. These problems are believed to be caused by lake eutrophication due to human activities and accidental water level drops, which could reach the light at the bottom of the lakes, facilitating waterweed growth (Shiga Prefecture, 2009).
On the other hand, waterweeds play a critical role in the Lake Biwa environment and for its surrounding residents. Because waterweeds are closer to the water surface than to the lake bottom, they can provide a space where animals and plants can settle in the lake with leaves and stems and where light and nutrients can be used efficiently (Maruno & Hamabata, 2016). Accordingly, the moderate growth of waterweeds leads to increased biodiversity and water purification for absorbing nutrients in the water.
Then, waterweeds have been systematically harvested using dedicated reaping ships to restore a suitable growth environment for native species of the 1930s-1950s in Shiga Prefecture. A prefectural consignment agency composts the harvested waterweeds. The compost is distributed to the residents inhabiting the lake surroundings for free to encourage the effective utilization of waterweeds (Ban et al., 2019;Osono et al., 2015).
Composting is an aerobic process in which microorganisms break down complex degradable substances into organic and inorganic byproducts (Toledo et al., 2018). Composting converts organic waste into products that can be safely and beneficially used as an organic fertilizer and a soil amendment (Yu et al., 2019). Adding compost to cultivated soil can increase crop productivity and organic matter content in the soil because the composted material has sufficient nutrients and the presence of organic matter that promotes plant growth (Pane et al., 2014). Composts can be regarded as a valuable material for raising the pH of acidic soil (Ch'ng et al., 2014) and improving the physical, chemical, and biochemical properties of soil (Lakhdar et al., 2009). Additionally, using compost in agriculture benefits greenhouse gas reduction (Razza et al., 2018).
In the past, the waterweed compost was constantly used as a valuable fertilizer and soil amendment and supported local agriculture. Even now, the effective use of the waterweed compost is crucial from the viewpoint of improving the environment of Lake Biwa and promoting the recycling of local resources (Hiratsuka et al., 2006). Still, scientific information on the resource value as a fertilizer or a soil amendment is required to revitalize the use of the waterweed compost and realize sustainable agriculture. However, the effective utilization of that in agriculture remains poorly understood in some ecological studies of the waterweed compost of Lake Biwa (Matsuoka et al., 2018;Matsuoka, Fujinaga, et al., 2020;Osono et al., 2015). Therefore, in this study, we conducted a pot experiment using Komatsuna (Brassica rapa var. perviridis) with the waterweed compost to assess its certified characteristics in agriculture, measuring its benefits in the growth of Komatsuna.

Waterweed compost
Waterweeds harvested at Lake Biwa in Shiga Prefecture were piled in Omi-Hachiman City, Shiga Prefecture, Japan, for at least 2 years while occasionally inverting. The waterweed compost was sieved through 2-mm meshes and used for a pot experiment. In this experiment, we used the waterweed compost distributed by Shiga Prefecture in 2019. Table 1 shows the properties of the waterweed compost analyzed according to the official fertilizer analysis method. We have confirmed that the characteristics do not change significantly, even when compared yearly (Supplementary Table S1).

Compost maturity
The maturity evaluation of a given compost can be assessed not by a single property but by many parameters (Mahapatra et al., 2022). Then, the waterweed compost maturity was evaluated by conducting a germination test and assessing the nitrifying activity of the waterweed compost in this study.
Five grams of the waterweed compost was added to 100 mL of deionized water and incubated at 60°C for 3 h. The solution was then filtered using filter paper (Fujiwara, 1985). For the germination test, two sheets of filter paper were laid on each of the two Petri dishes with a diameter of 90 mm. A total of 10 mL of the filtrate was added to a Petri dish. As a control, 10 mL of distilled water was added to the other Petri dish. Fifty gains of Komatsuna seeds (Sakata Seed, Japan) were placed on filter paper. Then, the Petri dishes were incubated at 28°C in the dark for 2 days and checked for germination. The filtrate was determined for nitrate and ammonium ion contents using a reflectometer (RQflex 20, Merck, Germany) to evaluate its nitrifying activity.

Cultivation test
The effect of the waterweed compost as an organic fertilizer was verified using a Komatsuna cultivation test. The waterweed compost was mixed with Akadama soil. The ratio of the waterweed compost to the soil was 0% (w w −1 ; i.e. Akadama soil only), 5% (w w −1 ), 10% (w w −1 ), 20% (w w −1 ), 30% (w w −1 ), and 50% (w w −1 ), and the mixed soil was filled in a black polypot with a diameter of 12 cm (WC plot). Chemical fertilizer (urea as nitrogen fertilizer, lime superphosphate as phosphoric acid fertilizer, and potassium sulfate as potassium fertilizer) was applied to Akadama soil as a basal fertilizer with the same quantity of N, P 2 O 5 , and K 2 O contents of each waterweed compost. The soil was filled in the same polypot (CF plot). A cultivation test of Komatsuna was conducted by changing the content of the waterweed compost in the presence of chemical fertilizer to verify the effect of the waterweed compost as a soil amendment. Chemical fertilizer (N:P 2 O 5 :K 2 O = 8:8:8) was evenly added to Akadama soil with the recommended fertilization amount (150 g m −2 ). The waterweed compost was mixed with the fertilized Akadama soil so that the ratio of the waterweed compost to the soil was 0% (w w −1 ; i.e. containing chemical fertilizer only), 5% (w w −1 ), 10% (w w −1 ), 20% (w w −1 ), 30% (w w −1 ), and 50% (w w −1 ). The mixed soil was filled in a black polypot with a diameter of 12 cm (WC + CF plot).
Komatsuna was cultivated for 4 weeks in all cultivation tests. After the end of the cultivation period, the aboveground part and the soil were sampled. Following the measurement of fresh weight, it was dried at 70°C for 3 days to measure the dry weight and supplied for component analyses. The sampled soil was supplied for measurement of the amount of soil microorganisms. All cultivation tests were conducted in triplicate in a glass greenhouse at Ryukoku University in Otsu City, Shiga Prefecture, Japan.

Component analysis
The dried plant sample was powdered, wet ashed, and analyzed for inorganic components. Approximately 100 mg of powdered dry sample was placed in a polyethylene tube with a lid (DigiTUBEs; GL Sciences, Japan). A total of 3 mL of nitric acid was added to the tube. The tube was incubated overnight at room temperature and set in a decomposition apparatus (DigiPREP MS; GL Sciences, Japan). After decomposition at 90°C for 140 min, the lid was opened, and the tube was continued to be heated at 100°C for 30 min. After adding 2 mL of 30% hydrogen peroxide, the lid was shut again, and the tube was further heated at 90°C. After 30 min, the lid was opened, and the tube was heated to 90°C. When the solution volume in the tube was decreased to 500-700 μL, the apparatus was turned off. After cooling, the solution was diluted to 10 mL by 0.08 N nitric acid and passed through a 0.45-μm filter. The inorganic components of the filtrate were quantified using ICP-AES (iCAP7400; Thermo Scientific, USA). The total carbon and the total nitrogen contents were determined using a dry powder sample in an automatic elemental analyzer (VarioMax Cube; Elementar, Germany).

Measurement of the amount of soil microorganisms
The ATP content in the soil was determined immediately after cultivation to measure the relative amount of soil microorganisms (Contin et al., 2001). Two grams of the cultivation soil were quickly mixed with 200 mL of deionized water and shaken vigorously for 2 min. After leaving to stand for 2 min, 100 μL of supernatant was absorbed into LuciPack Pen (Kikkoman Biochemifa, Japan) and quantified for ATP content with Lumitester Smart (Kikkoman Biochemifa, Japan). The unit of light emission is expressed in a relative light unit.

Soil solution and DNA analysis
Komatsuna was cultivated using a 1/5000a Wagner pot for the soil solution and DNA collection. Each Akadama soil mixed with the recommended fertilization amount of chemical fertilizer (CF soil), or 20% (w w −1 ) of the waterweed compost (WC20 soil), or 20% (w w −1 ) of the waterweed compost and the recommended fertilization amount of chemical fertilizer (WC20 + CF soil) put into a 1/5000a Wagner pot. Komatsuna was cultivated in these three different soils for 4 weeks similar to the cultivation method above. The soil solution was collected weekly for the duration of cultivation using a soil solution collector (DIK-300B; Daiki, Japan) immediately after watering. Nitrate ion and ammonium ion in the solution were quantified using a reflectometer (RQflex 20, Merck, Germany).
The soil attached to the roots of Komatsuna was gently pulled out and collected. The soil was immediately stored frozen (−20°C) and thawed at room temperature for use when extracting DNA. According to the manufacturer's instructions, DNA was extracted from the soil using a DNA extraction kit (DNeasy PowerSoil Pro Kits; QIAGEN, Netherlands). Bacterial flora analysis was commissioned by an analysis supplier (Biological Research Institute, Japan). Diversity analysis was performed using QIIME 2 (Bolyen et al., 2019). The complexity of species diversity in the sample was analyzed using alpha diversity via two indices, including Chao1 and Shannon indices. Chao1 and Shannon indices were used to identify the community richness and evenness, respectively. The differences of samples in species complexity were evaluated using beta diversity analysis. Beta diversity on unweighted UniFrac distance was calculated. Clustering analysis was performed by principal coordinate analysis (PCoA), which was conducted to get principal coordinates from complex multidimensional data. Predictive metagenomic analysis was performed using PICRUSt2 (Douglas et al., 2020).

Statistics
All statistical analyses were performed using Excel Statistics (BellCurve, Japan). Student's t-test was used to compare the two treatments. Multiple comparisons were performed using the Tukey -Kramer test.

Maturity of the waterweed compost
As a result of the germination test, it was confirmed that the germination rate in the waterweed compost extract was 100% and that the root length and morphology were equivalent to those of the control (deionized water). In addition, the concentrations of nitrate and ammonium ions in the waterweed compost extract were 80 and 0.3 mg L −1 , respectively, suggesting that it was sufficiently nitrified. From these parameters, it was confirmed that the waterweed compost used in this study was completely matured.

Effect of the waterweed compost as an organic fertilizer
Komatsuna was cultivated by changing the input amount of the waterweed compost to verify its effects as an organic fertilizer. For comparison, chemical fertilizers were added to the soil so that the same amounts of N, P 2 O 5 , and K 2 O were contained in the WC plot, and Komatsuna was cultivated in the soil (CF plot). The growth of Komatsuna was markedly promoted as the amount of the waterweed compost input increased. Particularly, in over 20% (w w −1 ) of the waterweed compost, a significant increase was observed compared with that in the CF plot ( Figure 1A). However, in the WC plot with a 50% (w w −1 ) addition, although the growth was more than twice as much as that in the CF plot, morphological abnormalities were observed; i.e. the leaves somewhat shrink and the stems were curved (Supplementary Fig. S1). In the CF plot, growth tended to increase depending on the amount of chemical fertilizer. Still, the amount of chemical fertilizer input equivalent to more than 5% (w w −1 ) of the waterweed compost did not indicate significant differences. From the above, the waterweed compost functions as an excellent organic fertilizer, and the effective minimum input amount is 20% (w w −1 ).
The components contained in Komatsuna cultivated under each nutritional condition were measured. As a result of quantifying the total carbon content and total nitrogen content, the C/N ratios were at low values in the CF plot. Still, such values were significantly high in the WC plot, except for the 50% (w w −1 ) addition ( Figure 1B). Despite the growth promotion by adding the waterweed compost, the leaves were yellowed, demonstrating a nitrogen deficiency (Figure 3). When reaching 50% (w w −1 ) addition of the waterweed compost, no nitrogen deficiency symptom was observed. Still, it had an abnormal growth, as mentioned above ( Supplementary Fig. S1).
No significant changes were observed in potassium uptake except in the addition of the waterweed compost 50% (w w −1 ) ( Figure 1C). This is the result of abnormal growth. Regarding phosphorus, the amount of uptake increased significantly at 20% (w w −1 ) and above for the supply of the waterweed compost ( Figure 1D). Based on ATP measurements, soil microorganisms in the WC plot increased significantly compared with those in the CF plot ( Figure 1E).

Effect of the waterweed compost as a soil amendment
Komatsuna was cultivated by changing the input amount of the waterweed compost with the recommended amount of chemical fertilizer to verify the effect of the waterweed compost as a soil amendment. The recommended amount of chemical fertilizer (control) was almost the same as that of chemical fertilizer, equivalent to 20% (w w −1 ) of the waterweed compost. The dry weight also had almost the same value. Although the growth slowed down with the increase in the input amount in the CF and WC plots ( Figure 1A), under sufficient nutrition, the growth was synergistically promoted up to about four times the control as the amount of the waterweed compost input increased (Figures 2A and 3).
The C/N ratio was low in all treatments, indicating that nitrogen was sufficient ( Figure 2B). Both potassium and phosphorus contents increased up to three to four times the control according to the amount of waterweed compost input, showing significant differences at 20% (w w −1 ) and above ( Figures 2C, 2D). Soil microorganisms in the WC + CF plot increased almost like those in the WC plot ( Figure 2E). Therefore, it was confirmed that the waterweed compost functions as an effective soil amendment. In addition, the effective minimum input of the waterweed compost is 20% (w w −1 ) as an organic fertilizer.

Analysis of soil solution
Nitrate and ammonia concentrations were measured in the soil solution after adding the waterweed compost. As a result, since the waterweed compost originally contained a large amount of nitrate, high concentration of nitrate was detected in the soil solution immediately after adding the waterweed compost to the soil (WC20 soil and WC20 + CF soil). The nitrate concentration in WC20 soil decreased with time, but in WC20 + CF soil, it peaked 1 week after sowing and then decreased, keeping a high level. On the other hand, in CF soil, almost no nitrate was detected throughout the cultivation period ( Figure 4A). Ammonia peaked 1 and 2 weeks after sowing in WC20 + CF soil and CF soil, respectively. At the same time, ammonia was hardly detected in the WC20 soil during the cultivation period. The peak ammonia concentration in CF soil was several times higher than that in WC20 + CF soil ( Figure 4B), suggesting rapid oxidation of ammonia to nitrate in WC20 + CF soil.

Effects of the waterweed compost on soil bacteria
DNA extracted from the soil attached roots of Komatsuna cultivated in CF soil, WC20 soil and WC20 + CF soil was supplied for bacterial flora analysis. As a result of the analysis, community richness and evenness in WC20 + CF soil were significantly higher than those in CF soil and WC20 soil (Table 2). Moreover, PCoA analysis (unweighted UniFrac distance) showed that the samples in WC20 + CF soil tended to cluster together, and the samples in CF soil and WC20 soil tended to cluster together (PC1, which explained 41.27% of the variation in the community) ( Figure 5). These results indicate that the addition of the waterweed compost with chemical fertilizer led to a significant diversification of soil bacteria.

Discussion
The waterweed compost has been a valuable agricultural material used for a long time in the area around Lake Biwa. However, it has been replaced by chemical fertilizers and other cheap and readily available composts (Hiratsuka et al., 2006). Scientifically evaluating the waterweed compost and encouraging its use is essential for realizing sustainable agriculture.
The maturity of the waterweed compost used in this study was investigated because it is helpful for crops only when the compost is sufficiently matured (Chen et al., 2018). As a result, it became clear that the waterweed compost used in this study was completely matured. In Shiga Prefecture, harvested waterweeds are piled up in the open field and composted by natural fermentation with occasional stirring. Empirically, it takes a long period of 2-3 years for the waterweeds to compost until it looks like soil. This is mainly due to the inability to cover the cost of composting. If the value of the waterweed compost as a fertilizer is recognized and if it becomes used commercially, it will be possible to   build composting facilities where high-quality compost will be produced in a short time. Although the amount and type of waterweeds harvested vary yearly, the composition of the resulting waterweed compost does not change significantly (Supplementary Table S1). Therefore, there seems to be no relationship between the type of waterweeds and the properties of the waterweed compost. This means that the waterweed compost could be supplied stably in the future. In this study, it was shown that the waterweed compost made with waterweeds from Lake Biwa is significantly effective, both as an organic fertilizer and as a soil amendment in the cultivation of Komatsuna. Akadama soil was used as a soil containing no fertilizer components in order to simplify our experimental system. The Akadama soil has a high ability to adsorb phosphate and is likely to cause phosphate deficiency symptoms in crops (Kitou et al., 2009). The suppression of growth in the CF plot may also be due to the inability of phosphate absorption in this experiment ( Figure 1D). However, the presence of the waterweed compost removed the restrictions on phosphate and promoted growth. This is thought to be the result of the high expression of alkaline phosphatase in soil and the promotion of phosphorus absorption by fertilizing the waterweed compost, as suggested by previous studies (Matsuoka, Fujinaga, et al., 2020). It is presumed that this is why the growth difference between the CF and WC plots is remarkable. As a result, this cultivation test made the potential of the waterweed compost stand out.
The phenomenon of growth promotion under even nitrogen deficiency in the WC plot cannot be explained from the viewpoint of phosphate acquisition. In general, nitrogen nutrition and plant growth are positively correlated (Zhao et al., 2005), so a nitrogen deficiency should suppress growth remarkably. However, the biomass was much larger than that in the CF plot, despite the nitrogen deficiency in the WC plot. This phenomenon is thought to be due to the growth-promoting effect that does not depend on nitrogen nutrition.
A meta-analysis reported that soil physicochemical properties were the most influential factor on crop yield due to the application of organic soil amendments (Shu et al., 2022). It is possible that the addition of the waterweed compost improved hardness, water-holding capacity, air permeability, and fertility-holding capacity of the soil, thereby promoting plant growth even if nitrogen nutrition was not enough. Alternatively, waterweed compost may prevent salinity stress on Komatsuna, which is susceptible to injury from excess salt. In addition, the waterweed compost may contain the substances similar to biostimulants which promote plant growth (Calvo et al., 2014). Since biostimulants have been reported in many seaweeds (Battacharyya et al., 2015), waterweed compost derived from the same aquatic plants may contain them as well. If the existence of biostimulants in the waterweed compost becomes clear, its value will be re-evaluated as a third agricultural material in addition to its function as an organic fertilizer and a soil amendment.
In the WC and WC + CF plots, a significant increase in the amount of microorganisms was confirmed according to the amount of waterweed compost input ( Figures 1E, 2E). In this study, the amount of soil microorganisms was evaluated based on the amount of ATP in the soil. Therefore, it is not possible to distinguish between bacteria and fungi. It can be interpreted that the increase in the amount of soil microorganisms is the result of active photosynthesis due to the development of the aboveground part and promoted the secretion of organic substances from the roots. However, although plant growth in the WC + CF plot was about twice as large as that in the WC plot, the amount of soil microorganisms was almost the same. This suggests that soil microorganisms may have increased because of the direct effect of the introduction of the waterweed compost.
As a result of the bacterial flora analysis, it was shown that the application of the waterweed compost Figure 5. Results of PCoA. The x-axis represents one principal component, the y-axis represents another component, and the percentage represents the contribution of the principal component to the sample difference. Each node represents a sample, and the samples from same treatment are represented by the same symbol. Black open circles represent CF, Grey closed circles represent WC20 and Blank closed circles represent WC20 + CF.
increased the amount of soil microorganisms and changed the soil bacteria composition. Interestingly, the application of the waterweed compost with chemical fertilizer caused significant diversification of the soil bacteria (Table 2 and Figure 5). It has been reported that diversification of soil microorganisms has a positive correlation with an increase in crop yield due to disease suppression, nitrogen cycling, and so on Mazzola, 2004;Shu et al., 2022). Similar effects were confirmed in our study; i.e. the dry weight of Komatsuna in the WC + CF plot, where soil bacteria were diversified, was about twice that in the WC plot ( Figures. 1A, 2A).
Predictive metagenomic analysis using PICRUSt2 predicted multiple statistically significant differences in soil microbial function. We noted that the reducing TCA cycle, i.e. a carbon dioxide fixation pathway found in autotrophic eubacteria and archaea (Smith & Morowitz, 2004), was significantly more active in the WC20 and WC20 + CF soil than in the CF soil (Supplementary Table S2). This implies that nitrifying bacteria, which are chemosynthetic, may proliferate and activate with the addition of the waterweed compost. It can be expected that the effect will be further increased by coexisting chemical fertilizer with the waterweed compost (Supplementary Table S2). Analysis of the soil solution showed a high nitrate concentration in the WC20 + CF soil and the peak of nitrate and ammonia concentration coincided (Figure 4). It can be confirmed that rapid oxidation of ammonia has occurred in the WC20 + CF soil.
In general, organic farming, in which only organic fertilizers are used, is attracting attention. However, our results suggest that using organic and chemical fertilizers in combination makes it realistic to achieve a high yield while controlling the amount of chemical fertilizers used. Organic farming is recommended as an environment-conserving agriculture, but careful discussion based on scientific facts will be necessary in the future.
Since waterweeds have a high carbon dioxide absorption capacity, it is crucial to study their utilization from the viewpoint of carbon neutrality. Although there is research on using waterweeds as an energy source by methane fermentation (Koyama et al., 2015), its agricultural use has long been promising. This study revealed possibilities for the waterweed compost: growthpromoting and diversification of soil microorganisms. It is also possible to realize the resource cycle between the water and land systems for a sustainable society. Further research is needed as the application method of waterweed compost has not been well established. If such a method is established, it is believed that it can greatly contribute to sustainable agriculture.