Vermicomposting as a Sustainable Option for Managing Biomass of the Invasive Tree Acacia dealbata Link

: The tree Acacia dealbata is native to Australia but has become invasive in many parts of the world thanks to its N-fixing capacity and to the allelopathic compounds present in its biomass. We conducted a pilot-scale study to assess the potential conversion of A. dealbata biomass by vermicomposting via the earthworm Eisenia andrei . The flowering aerial A. dealbata biomass was shredded and placed in a vermireactor under greenhouse conditions for 56 days. The vermicomposted material was sampled every two weeks to analyse its biological and chemical parameters. The phytotoxicity of the fresh A. dealbata material and vermicompost was assessed via an ecotoxicological test with Lepidium sativum seeds. The activity of the earthworms caused strong modifications of the properties of the processed material: the electric conductivity, basal respiration, and organic matter content were reduced, whereas the concentrations of other elements such as N, P, or Zn increased. The earthworm biomass increased steadily until day 42 and then decreased, probably due to the depletion of labile organic matter during the initial stages of vermicomposting. The fresh A. dealbata material reduced the germination and radicle elongation of L. sativum , whereas vermicompost showed the same values as control. The produced vermicompost was an organic fertiliser rich in N and was not phytotoxic. Vermicomposting provides an opportunity to create a new value chain for the control of the invasive tree A. dealbata .


Introduction
Acacia dealbata Link (Fabaceae) is native to Victoria, New South Wales, and Tasmania (Australia), where it can grow as a shrub or tree, reaching heights of up to 30 m [1]. A. dealbata is a fast-growing species with showy, scented flowers. It is able to develop in nutrient-poor soils thank to its ability to fix atmospheric nitrogen [2]. During the nineteenth century, A. dealbata was introduced in Europe, India, South Africa, and South and North America for multiple purposes: ornamental use, wood and tannins production, dune stabilization, or for the revegetation of bare land [3][4][5][6][7]. After its introduction in new areas, A. dealbata has expanded its range and is currently considered an invasive weed in SW Europe (Spain, Portugal, France, and Italy), South Africa (Cape floristic region), and areas with a Mediterranean climate in Chile [3,8]. The invasive behaviour of A. dealbata has been explained by the presence of several traits: a tolerance for changing soil conditions (including a capacity for nitrogen fixation and high water consumption), the ability to take advantage of environmental disturbance, phenotypic plasticity, vegetative reproduction (by the presence of rhizomes), fire tolerance (with the capacity to resprout and germinate profusely after forest fires), and an allelopathic potential [5,6,9]. Several compounds with phytotoxic effect have been found in A. dealbata, such as alkaloids, cyanogenic glycosides, terpenes, flavonoids, and tannins [7]. Although these compounds are mainly present during the flowering period [10], some studies have indicated a phytotoxic effect throughout the entire phenological cycle [7,11]. In spite of this recognised phytotoxicity of A. dealbata's biomass, recent studies indicate that allelopathy may be a secondary factor in the success of this species, whereas modifications in microhabitat induced by A. dealbata after its colonisation of a site constitute the main driver affecting seedling establishment in the European non-native range [12].
In Galicia (NW Spain), A. dealbata has extensively invaded natural forests, abandoned arable land, and water courses, causing an important impact on the native flora and even impacting protected areas [5,9]. According to these impacts, this species has been included since 2013 in the Spanish Catalog of Invasive Alien Species [13]. The inclusion of a species in this Catalog entails, among other measures, a generic prohibition of the possession, transport, traffic, and trade of live specimens, their remains, or propagules that could survive or reproduce, as well as the preparation of a control and eradication program for those species considered priority risks [14].
Although some biological control measures have been proposed for A. dealbata and other Acacia species in Europe [3,5] and tested in South Africa [15], the main strategy used is the mechanical removal of the aerial parts, sometimes in combination with the use of herbicides to reduce resprouting [13,16]. However, this process is expensive and generates biomasses with potentially negative effects on the flora and soils due to the presence of allelopathic compounds.
Recently, some authors have proposed the creation of value chains from A. dealbata, either by its use as biofuel or as a source of valuable compounds and biochemicals, as a way to improve the control and eradication of this invasive species [17,18]. Here, we propose an alternative: the conversion of A. dealbata biomass into a fertiliser by vermicomposting.
Vermicomposting is the managed processing of organic waste by the synergistic action of earthworms and microbial communities [6]. The enzymes involved in the biochemical decomposition of organic matter are produced by microorganisms, and earthworms accelerate this decomposition by comminution and mixing of plant debris, which boosts the activity of bacteria and saprophytic fungi. Vermicomposting is based on the use of epigeic earthworms, which are litter dwellers that live in organic horizons [19]. In spite of the high diversity of terrestrial earthworms, with around 4000 species described to date [20], only four species are routinely used in vermicomposting facilities: Eisenia andrei (Bouché), E. fetida (Savigny), Perionyx excavatus (Perrier), and Eudrilus eugeniae (Kinberg) [21]. Previous studies have demonstrated the feasibility of vermicomposting for converting organic waste, such as sewage sludge, paper waste, urban residues, and animal waste (see [19,22] for reviews), into organic fertilisers. Vermicomposting has also been shown to be effective for processing plant wastes rich in phytotoxic compounds, such as grape marc, spent coffee grounds, and Scotch broom biomass [23][24][25].
In this research, we explored the potential of vermicomposting as a sustainable option for the management of A. dealbata biomass and its conversion into an organic fertiliser. We had two objectives: (i) to estimate whether the earthworm E. andrei was able to degrade the biomass of A. dealbata, rich in secondary compounds, and (ii) to assess the potential phytotoxic risk of the obtained vermicompost. For this purpose, we conducted a pilot-scale vermicomposting trial and combined the analysis of biochemical parameters throughout the process with an ecotoxicological bioassay of the phytotoxicity of the final product.

Plant Material
Several young (around 5 m in height) silver wattle trees (Acacia dealbata Link) were cut down in a forest near the University of Vigo (NW Spain) in spring, when the trees were flowering. Young branches, leaves, and flowers were manually collected from those fallen trees. The branches were chopped into smaller pieces of 3-6 cm, whereas the flowers and leaves were left intact. The fresh weight of the total sample was 120 kg.

Vermicomposting Setup
The collected material was processed in a rectangular, metal, pilot-scale vermireactor (length 4 m × width 1.5 m wide × height 1 m) held in a greenhouse without temperature control. Before the material was added to the vermireactor, it contained a layer of vermicompost (12 cm height), which acted as a bed for the earthworms (Eisenia andrei). The initial earthworm density in the vermireactor was 344 ± 10 individuals per m −2 , with a mean biomass of 99.4 ± 4.5 g m −2 . The vermicompost layer was covered by a plastic mesh (5 mm mesh size), and the tree material was added to the vermireactor in a layer (12 cm) on top of the plastic mesh. The plastic mesh allowed earthworms to move between the layers but prevented mixing of the processed material and the vermicompost bedding. It also simplified sampling of the processed material during vermicomposting.

Data Collection
The density and biomass of the earthworm population was determined periodically by collecting 10 samples (five from above and five from below the plastic mesh) of the material in the vermireactor with a core sampler (7.5 cm diameter and 12 cm height) at each sampling time. Sampling was conducted every 14 days during the vermicomposting trial and ended after 56 days. In total, 50 samples were collected. At the end of the experiment, a total of 21 kg (fresh weight) of vermicompost had been produced.
In order to estimate the evolution of the physico-chemical properties of the substrate along the vermicomposting process, we divided the surface of the vermireactor into five sections and removed two subsamples (10 g) at random from each section at the beginning of the experiment (day 0) and after 14, 28, 42, and 56 days of vermicomposting. The two subsamples from each section were bulked and stored in plastic bags at −80 °C until analysis. In total, 25 samples of substrate were collected and analysed.

Physico-Chemical Analyses
The moisture content was assessed after drying the samples for 24 h at 105 °C. The organic matter (OM) content was determined from the weight loss after combustion in a Carbolite CWF 1000 muffle furnace at 550 °C for 5 h. The pH and the electrical conductivity (EC) were measured in aqueous extracts (1:10 w/v) with a Crison MicropH 2000 pH meter and a Crison CM35 conductivity meter, respectively. Samples were ovendried at 60 °C. Total C and N content were determined in dry samples in a Carlo Erba EA 1108 CHNS-O 1500 C/N analyser. The total P, K, Ca, Mg, S, Fe, Mn, B, and Mo concentrations were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) in dry, ground samples that were subjected to microwaveassisted acid digestion. The cellulose, hemicellulose, and lignin content were assessed by detergent fibre methods [26] using the FibreBag System (Gerhardt, Königswinter, Germany).

Microbiological Analyses
Basal respiration was estimated from CO2 production. Subsamples of the plant material and vermicompost (5 g fresh weight) were placed in 100 mL glass vessels, which were then hermetically sealed and incubated at 22 °C for 6 h. The CO2 produced from the sample was trapped in 0.02 M NaOH. CO2 production was quantified by titration with 0.01 M HCl to a phenolphthalein endpoint, after addition of excess BaCl2. Microbial biomass carbon (Cmic) and microbial biomass nitrogen (Nmic) were determined by the fumigation-extraction method [27,28] with moist subsamples (5 g fresh weight).

Phytotoxicity Assay
A germination and radicle elongation test was performed with Lepidium sativum L. to estimate the potential phytotoxicity of A. dealbata biomass (collected at t = 0) and the vermicompost (collected at the end of the experiment; t = 56). Briefly, 20 L. sativum seeds were placed on a filter paper in a sterile 9 mm diameter Petri dish into which 5 mL of one of the following solutions had already been added: distilled water (control, C), 5% w/v of A. dealbata fresh biomass (AD5), 10% w/v of A. dealbata fresh biomass (AD10), 5% w/v of vermicompost (VC5), or 10% w/v of vermicompost (VC10). To prepare the AD5, AD10, VC5, and VC10 solutions, the relevant material was suspended in water and filtered after shaking for 1 h. Each treatment was prepared in triplicate, yielding a total of 15 Petri dishes and 300 seeds. After the seeds were sown, the Petri dishes were sealed with parafilm to prevent desiccation. The plates were incubated for 7 days at 20 °C in dark conditions. The plates were then collected and the number of germinated seeds in each plate was recorded. The radicle length of all germinated seeds was also recorded.

Statistical Analyses
The effect of time on the earthworm density and biomass in the vermireactor was estimated by a one-way ANOVA, followed by post hoc Tukey tests. Vermicompost chemical and microbiological parameters (except the content of fibers) were analysed by Principal Component Analysis (PCA) and a reduced number of Principal Components (PCs) that captured most of the variability in the data were extracted. The PCA was based on a correlation matrix, which is appropriate for data with different measurement scales. Scores of vermicompost samples on PCs with eigenvalues higher than 1 were retained and correlated with earthworm density and biomass by the Pearson correlation index. The phytotoxic effect of A. dealbata biomass and vermicompost on germination was assessed by a one-way ANOVA, and the differences in radicle length were assessed by a nested ANOVA, with the replicate nested within the treatment. In case of significant differences, Tukey post hoc tests were used to determine where the differences lay. Where necessary, the data were transformed to meet ANOVA requirements. ANOVAs, PCA, and linear correlations were computed with SPSS (v. 15.0, SPSS Inc., Chicago, IL, USA).

Changes in the Properties of A. dealbata Vermicompost during Vermicomposting
The vermicompost produced by processing the A. dealbata biomass in the vermireactor for 56 days was slightly acidic (pH = 6.42), with total concentrations of N, P, and K of 39.2, 1.4, and 1.4 g kg −1 , respectively. The C/N ratio was 12.01, the concentrations of Cu and Zn were 27.8 and 270 mg kg −1 , and the concentrations of lignin, cellulose, and hemicellulose were 530.7, 188.1, and 57.4 g kg −1 , respectively. The values of the other chemical and microbiological parameters of A. dealbata vermicompost samples (averaged per sampling time) are included in Table A1 (Appendix A).
The application of a PCA analysis of the A. dealbata substrate and vermicompost allowed us to identify four Principal Components (PCs) that explained more than 90% of the total variance. The first principal component (PC1) alone explained 61.5% of the variance. The loadings (i.e., the correlations) of each parameter on the four PCs are presented in Table 1. Almost all parameters were correlated with PC1. The variables with positive correlations included the moisture content and the concentrations of several elements such as N, P, S, and others. By contrast, electrical conductivity, organic matter content, basal respiration, and the concentrations of C, B, and Cl were negatively correlated with PC1 (Table 1). Microbial C, pH, and potassium concentration were positively correlated with PC2. Microbial N was negatively correlated with PC3, whereas K was positively correlated with PC3. Only one parameter (Mo concentration) was correlated with PC4 (Table 1). After plotting the A. dealbata and vermicompost samples on the PC1-PC2 axes, we observed that the samples were arranged along PC1 as a function of time ( Figure 1): the PC1 scores for t0 samples were below −1.5, the scores for t14 samples were around −1, the scores for t28 and t42 were around 0.5, and the scores for t56 samples were higher than 1.
Considering these values and the parameters correlated with PC1, we interpreted that this axis provides information about the "vermicompost maturity". Regarding PC2, the samples from t0 and t56 had the lowest values, whereas the samples from the intermediate vermicomposting steps had the highest values. Given the correlation between microbial C biomass and PC2, we find that this axis indicated the "size of the bacterial community". The samples tended towards lower values of PC3 over the sampling time ( Figure A1, Appendix A), whereas the values of PC4 are similar for all sampling times except for the t28 samples, which yielded lower values ( Figure A1, Appendix A).

Changes in Earthworm Population
The population of earthworms in the vermireactor varied significantly throughout the experiment ( Table 2). The total earthworm density and biomass increased steadily from the beginning of the experiment. The maximum density (877 ± 34 individuals m −2 ) was reached on day 28 and remained stationary until day 42. The maximum earthworm biomass (404.2 ± 15.2 g m −2 ) was recorded on day 42. Both parameters then decreased until day 56 (Table 2). The earthworm population parameters (density and biomass) and principal component PC1 were highly significantly correlated. These parameters were also negatively correlated with PC4 ( Figure 2).

Phytotoxic Effects before and after Vermicomposting
The results show that the fresh A. dealbata material was phytotoxic. Thus, the germination percentage of L. sativum seeds in treatments AD5 and AD10 was around 80%, whereas the percentage of germination in the control was around 97% ( Table 3). The differences in root elongation between the control and treatments with A. dealbata were clearer: in the control, the average radicle length was slightly below 7 cm, whereas in AD5 and AD10 the average length was around 2 cm (Figure 3). By contrast, the treatments with mature vermicompost (VC5 and VC10) yielded similar germination percentages and radicle elongation rates as the control (Table 3 and Figure 3). Table 3. Variation in the germination percentage (after 7 days) of L. sativum seeds treated with water (C), A. dealbata extracts (AD5 and AD10), and vermicompost extracts (VC5 and VC10). The results of ANOVA (F and p-values) regarding the effects of the factor treatment are shown. The average ± standard deviation of germination percentage in each treatment are also shown. Different superscript letters indicate significant differences between treatments, according to Tukey post hoc tests.

Discussion
We performed a pilot-scale study to assess the feasibility of vermicomposting as a way of managing and valorising the biomass of the invasive tree A. dealbata. The earthworm Eisenia andrei was able to grow and reproduce in this substrate, with a sustained increase in the number of individuals and earthworm biomass until day 28, followed by a plateau between days 28 and 42. After that point, the number of earthworms and their total biomass decreased until the end of the experiment. This demographic pattern is typical of populations growing in closed systems (i.e., systems without any matter exchange with any part outside the system), where populations grow steadily until the point when the consumption of all the available resources or the accumulation of wastes provokes a decrease in the population (e.g., [29]). Our results show a continuous reduction in hemicellulose (from 13.3% to 5.7%) and cellulose (from 21.7% to 18.8%) and an increase in lignin (from 38.8% to 53.2%) in the substrate during vermicomposting, which indicates the consumption of the more labile organic fractions by the earthworms and an enrichment in recalcitrant, less-palatable materials. Similarly, high concentrations of lignin and polyphenols in the leaf litter of Acacia auriculiformis were suggested to be responsible for the high earthworm mortality during the vermicomposting of this litter [30]. Other researchers found that the increase in N compounds and the decrease in the C:N ratio (depending on the residues, changes of C:N ratio from 40 to 20) negatively affected the growth and reproduction of Eisenia fetida during the vermicomposting of diverse organic residues [31]. However, it seems that the C:N ratio is not the limiting factor for E. andrei, because (i) the population in our experiment grew while the C:N ratio decreased from 19.9 to 12, and (ii) in another vermicomposting study with E. andrei, the C:N ratio of the substrate stayed at values around 12 (11.5 at day 0 and 12.9 at day 42) and the earthworm population and biomass increased steadily with no signs of population stagnation [25].
In addition to the relative changes in the hemicellulose, cellulose, and lignin proportions mentioned above, the main trends observed during the vermicomposting of A. dealbata biomass included reductions in the EC; OM content; concentrations of B, C, and Cl; and basal respiration, and increases in moisture content and in the concentrations of N, Ca, Cu, Fe, Mg, Mn, P, S, and Zn. Similar processes have been documented during the vermicomposting of other substrates [23][24][25] and are mainly related to i) rapid C mineralisation throughout the vermicomposting process (especially of the most labile fractions of the cell walls), which involves the concentration of other elements, and ii) the reduction in the concentration of soluble ions due to leaching, precipitation into nonmobile salts, or immobilisation by microorganisms [24,32]. Other observed changes included an increase in pH and, especially, the microbial biomass of C during the intermediate stages of vermicomposting (day 14) and a decrease in these parameters until the final product. The observed variation in pH during vermicomposting (from acid values in the fresh material to more basic values in different vermicomposting stages) contrasts with the most common changes observed in the literature (progressive acidification during vermicomposting; see [33][34][35] and references therein), but is congruent with the changes observed during the aerobic composting of A. dealbata biomass [36]. While the acidification of the substrate during vermicomposting has been explained by the mineralisation of N and P and the formation of organic acids during the bioconversion of organic material [35], other authors have proposed that the increase in the pH in A. dealbata after composting was due to NH3 formation and the degradation of organic acids [36].
The final product was stabilised organic matter, as indicated by the decrease in the microbial biomass of C and the basal respiration, as well as the decrease and stabilisation of the C:N ratio. Moreover, the vermicompost obtained met the EU-criteria for solid, nitrogen-rich organic fertilisers [37], i.e., solid and of organic origin; an N concentration above 2.5%; an organic carbon content higher than 15%; and concentrations of Cu and Zn below 300 and 800 mg kg −1 , respectively.
A. dealbata is known to possess secondary compounds such as phenolics, triterpenes, or alkaloids with allelopathic effects [7,10,11]. Thus, the use of A. dealbata as an organic fertiliser could be hampered by the presence of these phytotoxic compounds in the vermicompost. We conducted an ecotoxicological test with Lepidium sativum, a plant which has been widely used in this kind of study (e.g., [38]). Our results indicate that vermicomposting eliminated the phytotoxicity of the fresh material. Moreover, radicle elongation was a parameter with a higher sensitivity than the seed germination percentage. Similar reductions in phytotoxicity were obtained after the composting of the vermicompost of diverse organic residues such as manure, biosolids, and urban or food wastes [39][40][41][42]. Although we did not quantify the secondary compounds present in the fresh A. dealbata biomass and the vermicompost, we assume that our results indicated that the phytotoxic compounds have been degraded during the vermicomposting process. Accordingly, previous research found that vermicomposting with E. andrei totally degraded the polyphenols present in Scotch broom biomass [25] and reduced the concentrations of these phytotoxic compounds in grape marc [23]. Moreover, a recent study indicated that vermicompost derived from different types of organic waste can contain high amounts of different extracellular catalytic enzymes that are even capable of degrading organophosphorus pesticides [24].

Conclusions
We have performed a pilot-scale assay of vermicomposting as an alternative for the management of the invasive tree Acacia dealbata. We have found that E. andrei is able to grow and process the biomass of this tree and produce a stabilised material after 56 days. The obtained vermicompost meets the EU requirements for solid organic fertilisers. Our results also indicate that vermicomposting eliminated the phytotoxicity of A. dealbata biomass.
Some value chains have been proposed as a way to promote the control and eradication of the invasive tree A. dealbata. Overall, our findings indicate that vermicomposting with E. andrei is a feasible alternative for the conversion of this organic residue into a valuable fertiliser, thereby contributing to decreasing the impact of alien species on native biodiversity. Acknowledgments: The technical support provided by Hugo Martínez-Cordeiro, Alberto da Silva, and Victor González Sales is gratefully acknowledged.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.