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Article

Vermicast Analysis with the Earthworm Species Pheretima losbanosensis (Crassiclitellata: Megascolecidae): Bacterial Profiles for Potential Applications in Agriculture

by
Maria Reynalen F. Mapile
1,*,
Nonillon M. Aspe
2 and
Marie Christine M. Obusan
1
1
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Metro Manila, Philippines
2
College of Marine and Allied Sciences, Mindanao State University at Naawan, Naawan 9023, Misamis Oriental, Philippines
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(18), 10364; https://doi.org/10.3390/app131810364
Submission received: 2 August 2023 / Revised: 9 September 2023 / Accepted: 14 September 2023 / Published: 16 September 2023
(This article belongs to the Special Issue Current Trends in Composting and Vermicomposting Technologies and Their Utilization)
Browse Figures
Versions Notes

Abstract

:
In the Philippines, the use of non-native earthworm species in vermicomposting is popular. Given that the country is a vital geographical resource for earthworm diversity, the study of earthworm species to establish the potential of their vermicasts in agricultural applications is essential. In this study, the bacteria associated with the vermicasts of the recently described indigenous species, Pheretima losbanosensis, were investigated using next-generation sequencing, community-level physiological profiling, and NPK activity screening. The results showed diverse bacterial species belonging to the phyla Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Thermodesulfobacteria, and Verrucomicrobia. Higher diversity and carbon substrate utilization (p < 0.05) of amines and amides, phenolic compounds, polymers, and carboxylic and acetic acids were exhibited by the bacterial communities of P. losbanosensis compared to those of Eudrilus eugeniae. Likewise, bacteria (n = 25) isolated from P. losbanosensis vermicasts had higher nitrogen fixation and phosphate and potassium solubilization activities (p < 0.05) than the bacteria (n = 20) isolated from E. eugeniae vermicasts. Overall, our results indicate that the diverse bacterial communities inhabiting the vermicasts of P. losbanosensis have nutrient mineralization and carbon substrate utilization activities that may have applications in sustainable agriculture as a potential organic input to promote plant growth and improve soil substrate.

1. Introduction

Vermicomposting is the process of using earthworms for the degradation of materials, producing vermicasts with beneficial applications primarily in agriculture. Vermicasts excreted by earthworms were reported to be inhabited by diverse species of bacteria such as those obtained from Aporrectodea caliginosa Savigny, 1826 [1,2,3]; Aporrectodea trapezoides Dugés, 1828 [4]; Eisenia andrei Bouché, 1972 [5,6,7,8]; Eisenia fetida Savigny, 1826 [1,9,10]; Eudrilus eugeniae Kinberg, 1867 [9]; Libyodrilus violaceus Beddard, 1891 [11]; Lumbricus rubellus Hoffmeister, 1843 [12]; Lumbricus terrestris Linnaeus, 1758 [1,2]; Perionyx excavatus Perrier, 1872 [13]; and Pontoscolex corethrurus Müller, 1857 [14].
In general, vermicasts are characterized as having higher pH and moisture content than soil, along with greater enrichment by carbon, nitrogen, phosphorus, ammonium, polysaccharides, exchangeable cations (Ca2+, Mg2+, and K+), and humus [15,16,17,18]. The presence of higher nutrient content in vermicasts is associated with partial organic matter decomposition during gut passage, resulting in the conversion of these nutrients into available forms [19]. Water absorption, water holding capacity, enzymatic activities, microbial biomass (Cmic), metabolic quotient, cation exchange capacity, heavy fraction polysaccharide, soil organic matter, basal soil respiration, and base saturation were also higher in vermicasts [15,16,17,18,19,20]. Organic matter mineralization indicated by the Cmic/Corg ratio, wherein a higher ratio signifies higher activity, explains the involvement of vermicasts in nutrient mineralization resulting in the availability of more nutrients [19]. Earthworm vermicasts therefore offer favorable effects for improving soil properties and fertility, which in turn contribute to better plant growth and yield [18].
The application of vermicasts produced through vermicomposting provides numerous benefits to soil and plants [15,21], as shown by studies on cucumber, pepper, tomato, strawberry, marigold, and ornamental plants [22,23,24,25,26,27,28], to name a few. The yield and quality of cucumber or Cucumis sativus were improved upon the application of E. fetida vermicasts compared to fertilizers (organic and inorganic) [28]. Vermicasts produced by E. fetida, E. eugeniae, and Pe. excavatus were found to significantly increase the length of shoot and internode as well as the number of branches and leaves of Capsicum annuum when compared to plants treated with synthetic plant growth regulators [27]. Leaf areas, plant shoot biomass, and marketable weight of pepper and strawberries were increased when treated with vermicomposts [22,23]. An increase in the number of flowers and plant runners of strawberries was also evident. Shoot dry weights of tomato and marigold grown in vermicomposts of Eisenia spp. were comparable to those in a commercial horticultural medium (Metro-Mix 360) [24]. Improved length, initiation, number, and weight of the roots of an ornamental plant, Codiaeum variegatum, were observed when grown with vermicasts of Lampito mauritii Kinberg, 1867 and Perionyx ceylanensis Michaelsen, 1904 than the control peat moss [26]. These effects were associated with the high NPK content of earthworms’ vermicasts.
In spite of the known benefits of vermicasts, information on the physiological profiles and nutrient mineralization activities of their associated bacterial communities is lacking. In the Philippines, indigenous earthworm species are often neglected because vermicomposting usually involves the use of the non-native E. eugeniae since its introduction from Africa in the 1980s [29,30]. This epigeic species feeds on organic matter and occupies the surface litter, and is popular for its rapid growth, voracious feeding, and high tolerance to unfavorable conditions [7]. Currently, it is being promoted by the Philippines’ Department of Agriculture for its temperature preference of 25–30 °C, which is common in tropical countries [7,29]. Studies have proven the significant impact of E. eugeniae vermicasts as feed for Nile tilapia (Oreochromis niloticus) [31] and as fertilizer for lowland and upland rice [29,30,31]. However, E. eugeniae may pose a threat to native species being displaced. In this work, the diversity, physiological profiles, and nutrient mineralization activities of the bacteria associated with the vermicasts of P. losbanosensis Aspe and Obusan, 2023, a Philippine indigenous species, were assessed. Investigating the microbial activities in their vermicasts is important to establish their potential for sustainable agriculture. The Philippine archipelago is a vital geographical resource for indigenous earthworm diversity; however, there are no published studies and baseline data yet on their vermicasts and their associated bacteria as of the time of writing. To the best of our knowledge, this is the first report on the bacterial communities present in the vermicasts of an indigenous earthworm in the country, supporting the benefits of their utilization and potential application of their vermicasts as organic input in agriculture. Figure 1 illustrates the schematic diagram of this study.

2. Materials and Methods

2.1. Collection of Earthworm Samples

Adult P. losbanosensis earthworms were collected from Folia Tropica, a vermicompost facility in Los Baños, Laguna, Philippines (14.158882° N, 121.255919° E), that mass produces vermicasts for commercial purposes. Twenty adult earthworms were individually handpicked and placed in a polypropylene container [32]. Samples were immediately transported to the laboratory for processing. For vermicast collection, earthworms were kept in a container provided with aeration and moist sterile filter paper. After 12–15 h, fresh vermicasts were collected from the containers [33]. Adult E. eugeniae earthworms served as a control, collected from the vermicompost facility of the Diliman Environmental Management Office (DEMO), formerly known as the Task Force Solid Waste Management (TFSWM), University of the Philippines Diliman (UPD), Quezon City, Philippines (14°39′32.9″ N 121°04′02.1″ E).

2.2. Earthworm Species Morphological Diagnosis

2.2.1. Eudrilus Eugeniae

Body length 90–185 mm, tapering posteriorly, becoming thinly flattened at terminal end (Figure 2). Width 4–8 mm. Segments 161–211. Red-brown dorsum fading posteriorly; anterior with bright blue/green iridescent sheen from cuticle diffraction, ventrum beige, clitellum darker (sometimes lighter) than surroundings. Prostomium small, open epilobous. Dorsal pores, absent. Eight setae per segment from segment 2, closely paired. Clitellum covers segments 13, 14, 15–18, usually 13, 14–18, and interrupted ventrally. Male pores in 17 on tips of longitudinally grooved, tapering, eversible penis in large ventral chambers. Female pores combined with modified ‘spermathecal pores’, lateral, presetal in 14 as raised intrasegmental openings. Genital markings on 17 between male pores, faintly repeated in 18. Weakly muscular gizzard in v. Calciferous glands in x and xi. Intestine originates in or around xiv. Caeca absent. Male organs holandric with two large, unpaired sacs seen ventrally in x and xi, each containing a testis anteriorly and funnels posteriorly [34].

2.2.2. Pheretima losbanosensis

Large brown worms with adult size of 220–228 mm × 8–9 mm (Figure 3); equators pigmented; three pairs of spermathecal pores at 6/7–8/9; distance between spermathecal pores and between male pores 0.18 and 0.15 circumference apart ventrally, respectively; setae on vii and xx 26–36 and 56–66, respectively. Spermathecae paired in vii, viii, and ix, with nephridia on ducts; each spermatheca small with ovate ampulla, stout muscular duct, stalked diverticulum attached to duct near ampulla, terminating in ovate receptacles, stalks long and thin, some with convolutions; prostates small in xvii to xix; penis present [35].

2.3. DNA Extraction and Next-Generation Sequencing

Total DNA was extracted from 0.25 g of vermicasts using the NucleoSpin® Soil kit (Toronto, ON, Canada) following the manufacturer’s instructions. The concentration of extracted DNA was measured using a NanoDrop spectrophotometer and submitted to Macrogen for next-generation sequencing (NGS). The V3-V4 hypervariable region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform [4,36,37].
Raw DNA reads generated through NGS were analyzed. For assembly, paired-end reads were merged using FLASH (1.2.11) or Fast Length Adjustment of Short Reads. For pre-processing and clustering, CD-HIT-OTU was used for three-step clustering. Short reads were filtered out and extra-long tails were trimmed. Filtered reads were clustered at 100% identity. Chimeric reads were screened. The remaining representative reads from non-chimeric clusters were clustered into OTUs at 97% similarity at the species level [4,36,37]. Taxonomy assignment and diversity analysis were carried out using QIIME (Quantitative Insights into Microbial Ecology). Taxonomy assignment for the clustered OTUs was performed using QIIME-UCLUST to identify the taxonomic composition for each sample from phylum to species levels. Diversity analysis was carried out to determine the alpha diversity metrics including Chao1, Shannon, Simpson, and Good’s coverage.

2.4. Community-Level Physiological Profiling (CLPP) of Bacteria in Vermicasts

The physiological profiles of bacteria present in vermicasts of P. losbanosensis were determined [38]. Briefly, 1 g portions of vermicasts were suspended in 99 mL sterile distilled water for 20 min at 20 °C. After incubation for 30 min at 4 °C, 150 µL was inoculated into each well of Biolog EcoPlate with 31 carbon sources (β-methyl-D-glucoside, D-galactonic acid γ-lactone, L-arginine, pyruvic acid methyl ester, D-xylose, D-galacturonic acid, L-asparagine, Tween 40, i-erythritol, 2-hydroxy benzoic acid, L-phenylalanine, Tween 80, D-mannitol, 4-hydroxy benzoic acid, L-serine, α-cyclodextrin, N-acetyl-D-glucosamine, γ-amino butyric acid, L-threonine, glycogen, D-glucosaminic acid, itaconic acid, glycyl-L-glutamic acid, D-cellobiose, glucose-1-phosphate, α-keto butyric acid, phenylethylamine, α-D-lactose, D,L-α-glycerol phosphate, D-malic acid, putrescine) in three replications and incubated at 25 °C. Substrate utilization was indicated by the formation of a purple color upon the reduction of tetrazolium violet redox dye. Absorbance (OD) was measured at 590 nm every 24 h for 5 d. Average well-color development (AWCD), richness (R), and Shannon–Weaver index (H) were calculated using the absorbance data measured after 48 h, essential for microbial growth and color development. R was determined as the total number of oxidized carbon substrates with OD values ≥ 0.2. AWCD and H were calculated as follows, where ODi denotes optical density from each well and pi denotes the ratio of ODi to ∑ODi:
AWCD = ∑ ODi/31 H = −∑pi(lnpi)
All data analyses were performed using the statistical software R (version 4.3.0), specifically RStudio 5695.3.1.0 [39,40,41]. Analysis of variance (ANOVA) and post hoc Tukey’s HSD (honest significant difference) test at p ≤ 0.05 were performed to compare the AWCD, R, and H of the two earthworm species. Carbon substrate utilization patterns (CSUPs) were visualized using a heatmap generated by the gplots package.

2.5. Bacterial Isolation from Vermicasts

Bacteria were isolated from the vermicasts of P. losbanosensis through the pour plate method [1,42]. Briefly, 0.5 g portions of vermicasts were suspended in 2.5 mL sterile distilled water (1:5 ratio) and vortexed for a few minutes or until homogenized. Then, 1 mL of the homogenized sample was added to 9 mL of sterile distilled water to perform serial dilutions up to 10−3. From the last dilution, a 0.1 mL aliquot was inoculated onto nutrient agar (NA) with nystatin and incubated for 18–72 h at 37 °C and for 7 d in anaerobic conditions at room temperature. Colonies with distinct morphologies were selected and repeatedly sub-cultured for purification [1,11,32,42,43].

2.6. Bacterial Nutrient Mineralization Screening

Pure isolates were screened for nutrient mineralization, including nitrogen (N) fixation and phosphate (P) and potassium (K) solubilization, using nitrogen-free malate media supplemented with bromothymol blue (BTB), Pikovskaya’s agar (HiMedia M520), and Aleksandrow agar (HiMedia M1996), respectively. In each medium, 10 μL of fresh culture of bacteria from nutrient broth (NB) was spot-inoculated and incubated for 5 d at 37 °C. Daily observation was performed to check for a blue coloration zone indicating N fixation [44] and a clearing zone for P and K solubilization. The solubilization index (SI) was calculated based on the diameters of the colony and clearing zones [45,46,47]. ANOVA and post hoc Tukey’s HSD test at p ≤ 0.05 were performed to compare the coloration zone (N) and SI (P and K) values obtained using the statistical software R (version 4.3.0) through RStudio 5695.3.1.0.

3. Results and Discussion

3.1. Bacterial Community Profile of Vermicasts

The bacterial community targeted by the 16S V3-V4 region of the ribosomal DNA gene yielded 327,040 sequences from P. losbanosensis vermicasts (PL) and 200,984 sequences from E. eugeniae vermicasts (EE) (Table 1). After assembly, preprocessing, and clustering, 4734 and 6111 sequences were obtained and clustered into 280 and 133 OTUs from PL and EE, respectively (Table 2). The Good’s coverage of the samples was found to be 1.0 (PL) and 0.9997 (EE) (mean ± SD), which implies that most of the diversity was captured. The non-parametric Shannon index showed that more diverse species of bacteria were found in PL (6.6834) than in EE (4.2816).
Taxonomic assignment using QIIME-UCLUST resulted in a total of 12 bacterial phyla in the complete dataset, with 11 phyla in PL and 7 phyla in EE vermicasts (Figure 4). Major bacterial phyla identified were Bacteroidetes (41.6%) and Proteobacteria (35.3%). Proteobacteria is dominant in PL but Bacteroidetes dominate in EE. The other phyla include Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Thermodesulfobacteria, and Verrucomicrobia. Spirochaetes were not found in PL, while EE vermicasts were not inhabited by five phyla: Acidobacteria, Actinobacteria, Gemmatimonadetes, Nitrospirae, and Thermodesulfobacteria.
A total of 83 bacterial families were present in PL dominated by Vicinamibacteriaceae (12.0%), Cytophagales (8.72%), Thioprofundaceae (5.39%), Gammaproteobacteria (4.88%), Geodermatophilaceae (4.63%), Zoogloeaceae (4.14%), and Cytophagaceae (3.91%) (Figure 4). On the other hand, EE was inhabited by 37 families dominated by Flavobacteriaceae (53.56%), Cellvibrionaceae (9.11%), Moraxellaceae (6.50%), Caulobacteraceae (4.40%), and Pseudomonadaceae (3.80%). Genera Vicinamibacter (9.63%), Chryseolinea (8.72%), Thioprofundum (5.39%), Acidibacter (4.88%), Blastococcus (4.56%), and Azoarcus (4.14%) cover large parts of the 128 bacterial genera in PL, while Myroides (34.32%), Confluentibacter (11.34%), Cellvibrio (9.11%), Acinetobacter (6.35%), and Flavobacterium (4.17%) were relatively more abundant among the 50 genera in EE (Figure 5). In total, 153 and 68 species were present in PL and EE, respectively. Major bacterial species identified in PL were Vicinamibacter silvestris (9.63%), Chryseolinea serpens (8.72%), Thioprofundum lithotrophicum (5.32%), Acidibacter ferrireducens (4.88%), Blastococcus saxobsidens (4.56%), and Azoarcus olearius (4.06%). EE vermicasts were dominated by Myroides marinus (33.87%), Confluentibacter lentus (11.34%), Cellvibrio japonicus (7.15%), and Acinetobacter rudis (6.35%).

3.2. Physiological Profiles of Bacteria in Vermicasts

The AWCD and H indices of bacterial communities of PL (1.996 A590nm; 3.328 A590nm) were significantly higher (p < 0.05) than those of EE (1.042 A590nm; 3.216 A590nm) (Figure 6). The richness index showed that the bacterial communities in both vermicasts utilized all 31 carbon substrates. CSUPs (Figure 7 and Figure 8) of the two earthworm species showed that the bacterial communities in PL have significantly higher (p < 0.05) utilization of amines and amides (1.172 A590nm; phenylethylamine), phenolic compounds (1.088 A590nm; 2-hydroxy benzoic acid,) polymers (1.067 A590nm; tween 80 and alpha-cyclodextrin), and carboxylic and acetic acids (1.047 A590nm; D-galacturonic acid). On the other hand, significantly higher (p < 0.05) utilization of carbohydrates (1.160 A590nm; N-acetyl-D-glucosamine, D-mannitol, Beta-methyl-D-glucoside, and D-cellobiose) and amino acids (1.100 A590nm; L-asparagine and L-serine) were shown by the bacterial communities in EE.
Community-level physiological profiling allows the assessment of the functional diversity or metabolic potential of a microbial community as represented by carbon substrate utilization. The oxidative ability of a microbial community is indicated by the AWCD values in relation to the R index, signifying the number of oxidized substrates [48]. PL bacterial communities (1.996 A590nm) showed higher AWCD values denoting their oxidative ability compared to EE (1.796 A590nm), but both were able to oxidize all carbon substrates with an R value of 31. On the other hand, the H index measures the diversity of substrate utilization (DSU) in relation to the efficiency of microorganisms in substrate degradation [49]. Thus, a higher H value (3.356 A590nm) confirmed that bacterial communities present in PL were more efficient in the degradation of carbon substrates. These results show that bacterial communities associated with the vermicasts of P. losbanosensis with higher AWCD, R, and H indices possess higher functional diversity or metabolic potential [38].
To date, little is known about the physiological profiles of bacteria in earthworm vermicasts, except in the case of Lumbricidae earthworms such as Allolobophora chlorotica Savigny, 1826, A. caliginosa, and L. terrestris, which were reported to accelerate functional diversity in their vermicasts [50]. Their vermicasts had an AWCD value of 0.94 A590nm, which was significantly higher than the bulk soil and transitional zone (0.11–0.12 A590nm) of their burrow system. Moreover, groups of carbon substrates such as amino acids, carboxylic acids, carbohydrates, polymers, and miscellaneous compounds were favored in vermicasts and burrow systems as compared to bulk soil.

3.3. Nutrient Mineralization Activities of Bacteria in Vermicasts

A total of 25 bacterial isolates obtained from the vermicasts of P. losbanosensis and 20 isolates from E. eugeniae were subjected to plate-based screening assay of nutrient mineralization. Results showed 14 bacteria (PL: 8; EE: 6) capable of fixing N on nitrogen-free malate media, as indicated by blue-colored zones with maximum activity (p < 0.05) of 29 mm produced by isolate PL16 than the 9 mm produced by EE3 and EE4 (Figure 9 and Figure 10). Clearing zones produced by 18 isolates (PL: 10; EE: 8) confirmed P solubilization on Pikovskaya’s agar with isolate PL20 having a significantly higher (p < 0.05) SI value of 2.57 than EE5 (2.27) (Figure 9 and Figure 11). Clearing zones were also observed on Aleksandrow agar, showing the ability of seven isolates (PL: 3; EE: 4) to solubilize K with a significantly higher (p < 0.05) SI value of 3.3 produced by isolate PL4 than EE3 (2.67) (Figure 9 and Figure 12). Based on these data, bacteria isolated from the vermicasts produced by the indigenous earthworms showed higher plate-based NPK mineralization activities.
The presence of N-fixing bacteria (NFB) in earthworms’ vermicasts was previously reported in E. fetida, namely Azotobacter, Beijerinckia, and Derxia [10], and in E. eugeniae, including Aeromonas caviae and Bacillus xiamenensis [51]. The presence and activity of NFB in vermicasts have been confirmed to increase N mineralization in relation to the continuous casting activity of earthworms [52] as well as higher levels of microbial respiration, population, and enzyme activity [9]. Also, N mineralization involving NH4+ production is directly proportional to the size of vermicasts [53]. This was the case in Amynthas khami Thai, 1984, wherein an increase in NH4+ was observed from vermicasts with increasing size brought by fixation, denitrification, and remobilization in the microbial biomass. In contrast, the stabilization of vermicasts linked with a high rate of nitrification resulted in the decline in NH4+ content obtained from L. rubellus vermicasts [21]. Reduced ammonia content and a higher level of nitrate were also observed, wherein 96% of mineral N in fresh vermicasts is composed of ammonia converted to nitrate via nitrification [54].
Consistent with the results of the present study, vermicasts of E. eugeniae were reported to have P-solubilizing microbes such as Streptosporangium species [55] and A. caviae and B. xiamenensis [51]. Microbes capable of P solubilization were also isolated from other earthworm species such as Po. corethrurus vermicasts with acid phosphatase enzymes and rock phosphate-solubilizing microbes [14]. High phosphatase activity was also evident among L. mauritii, Pe. excavatus, Po. corethrurus, and Pheretima elongata Perrier, 1872, causing the release of more inorganic P through mineralization [56]. Among these, vermicasts of Pe. excavatus showed the highest phosphatase activity, indicative of its potential in organic matter mineralization. The passage of organic matter in the gut converts some P into a more available form, which is continued in vermicasts by P-solubilizing microbes [57].
Vermicomposting offers beneficial applications in agriculture, primarily in enhancing plant growth and soil substrate [15,16,17,18,19,20,21,22,23,24,25,26,27,28] owing to the diverse species of bacteria [1,2,3,4,5,6,7,8,9,10,11,12,13,14] and high nutrient content [15,16,17,18,19] present in vermicasts. Bacteria capable of nitrogen fixation and phosphate and potassium solubilization make the essential macronutrients readily available for plant uptake, resulting in improved quantity and quality of growth [15,16,17,18,19,20,21,22,23,24,25,26,27,28,58,59]. Microbial activities also influence soil restoration and augment soil structure, including increased porosity, water-holding capacity, and aggregate stability [60,61,62]. More than 170 vermicomposting systems were established in Cuba by 1994 replacing the need for 40 tons/hectare of livestock manure with 4 tons/hectare of vermicomposts. In Havana, Vivero Alamar Organoponico produces 300 tons of vermicasts per year. Production of a ton of organic fertilizer costs around USD 0.55, considerably cheaper than purchasing chemical fertilizer (40 USD/ton). In Sancti Spiritus, Finca de Casimiro established in 1993 also employed earthworms in the successful remediation of severely contaminated land used to be planted with tobacco [63]. This highlights the potential of vermicomposting as a promising alternative to chemical and synthetic inputs for sustainable agriculture. In the Philippines, the Los Baños Folia Tropica vermifacility in Laguna Province has been successfully culturing P. losbanosensis and mass-producing vermicasts for commercial purposes for more than a decade already. Based on the observations of Dr. Raymundo Lucero, who is managing the facility, indigenous earthworms in his facility have better cast yields and improved plant growth in comparison with E. eugeniae.
Interestingly, our results uncover the presence of diverse bacterial communities inhabiting the vermicasts of P. losbanosensis possessing nutrient mineralization and carbon substrate utilization activities, which are significantly higher than those of E. eugeniae. Bacterial species identified herein, including A. ferrireducens [64], Arboricoccus pini [65], A. olearius [66,67,68], Bradyrhizobium namibiense [69], Chitinophaga ginsengisegetis [70], Chitinophaga polysaccharea [71], Flavobacterium enshiense [72], Frankia discariae [73], Geobacter luticola [74], Herbaspirillum robiniae [75,76,77], Luteitalea pratensis [78], Methylosinus trichosporium [79], Nitrospira moscoviensis [80], Paludibaculum fermentans [78], Pelobacter propionicus [81], Pseudomonas oryzae [82], Roseiarcus fermentans [83], and Solirubrobacter ginsenosidimutans [84], were reported to be beneficial in the growth of plants like rice, grape, peanut, pine, blueberry, duckweed, legumes, beans, grass, and ornamental plants. Moreover, species of bacteria identified from the vermicasts of P. losbanosensis were also reported to be involved in soil bioremediation and the improvement of soil substrate such as Azoarcus tolulyticus [85,86,87], Caldimonas manganoxidans [88], Chitinophaga jiangningensis [89,90], Dehalogenimonas lykanthroporepellens [91], Desulfuromonas palmitatis [92,93], Ferruginibacter alkalilentus [94], Geobacter metallireducens [95], Geobacter sulfurreducens [96], Hyphomicrobium aestuarii [97], Immundisolibacter cernigliae [98], Methylibium petroleiphilum [99,100,101,102], Methylosinus trichosporium [103], Nocardioides islandensis [104], Phenylobacterium panacis [105], Povalibacter uvarum [106], Propionivibrio limicola [107], Pseudaminobacter manganicus [108], Steroidobacter denitrificans [109,110], Thermostilla marina [111], and Thiohalobacter thiocyanaticus [112]. These highlight the presence of beneficial bacteria in the vermicasts of P. losbanosensis that are involved in plant growth promotion, soil bioremediation, and soil substrate improvement, which are vital in sustainable agriculture practices.

4. Conclusions

Vermicasts of the recently described indigenous earthworm species, P. losbanosensis, were investigated and compared with E. eugeniae vermicasts. P. losbanosensis vermicasts harbor diverse bacterial communities comprising 153 different species belonging to the phyla Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, Thermodesulfobacteria, and Verrucomicrobia. The Shannon index confirmed that more diverse species of bacteria were found in P. losbanosensis. Physiological profiles of the bacterial communities in the vermicasts of P. losbanosensis were shown through the utilization of all 31 carbon substrates, primarily the amines and amides, phenolic compounds, polymers, and carboxylic and acetic acids, with higher AWCD and H index indicating higher functional diversity or metabolic potential. Among the 45 isolates, bacteria from the vermicasts of P. losbanosensis were found to be more efficient in plate-based NPK mineralization, showing the largest blue coloration zone, thus confirming nitrogen fixation and the highest phosphate and potassium solubilization index. Overall, this study unveiled that vermicasts produced by indigenous earthworms are better than those of non-native species in terms of having (1) more diverse bacterial communities, (2) higher functional diversity or metabolic potential, and (3) higher NPK mineralization activities. These results highlight the potential of P. losbanosensis vermicasts as an organic input to promote plant growth and improve soil substrate. Hence, Philippine earthworms should be further explored and harnessed for the production and utilization of vermicasts to mitigate the probable threats of invasive non-native species.

Author Contributions

Conceptualization, M.R.F.M., N.M.A. and M.C.M.O.; Methodology, validation, formal analysis, data curation, and writing—original draft preparation, M.R.F.M.; Writing—review and editing, M.R.F.M., N.M.A. and M.C.M.O.; Supervision and funding acquisition, M.C.M.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Commission on Higher Education (CHED) through the K to 12 DARE TO Research, grant number 9610145-499-416.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available as the content of reported tables and figures.

Acknowledgments

This research was conducted at the Microbial Ecology of Terrestrial and Aquatic Systems (METAS) Laboratory of the Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Schematic diagram of the study.
Figure 1. Schematic diagram of the study.
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Figure 2. Adult E. eugeniae earthworms collected from the vermicompost facility of the University of the Philippines Diliman, Quezon City, Philippines.
Figure 2. Adult E. eugeniae earthworms collected from the vermicompost facility of the University of the Philippines Diliman, Quezon City, Philippines.
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Figure 3. Adult P. losbanosensis earthworms collected from the vermicompost facility of Folia Tropica, Los Baños, Laguna, Philippines.
Figure 3. Adult P. losbanosensis earthworms collected from the vermicompost facility of Folia Tropica, Los Baños, Laguna, Philippines.
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Figure 4. Bacterial phyla (a) and dominant families (b) in P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME-UCLUST.
Figure 4. Bacterial phyla (a) and dominant families (b) in P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME-UCLUST.
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Figure 5. Dominant bacterial genera (a) and species (b) in P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME-UCLUST.
Figure 5. Dominant bacterial genera (a) and species (b) in P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME-UCLUST.
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Figure 6. Average well-color development (AWCD), richness (R), and Shannon–Weaver index (H) of carbon substrate utilization by P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
Figure 6. Average well-color development (AWCD), richness (R), and Shannon–Weaver index (H) of carbon substrate utilization by P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
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Figure 7. Carbon substrate utilization patterns (CSUPs) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
Figure 7. Carbon substrate utilization patterns (CSUPs) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
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Figure 8. Carbon substrate utilization patterns (CSUPs) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3).
Figure 8. Carbon substrate utilization patterns (CSUPs) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts’ bacterial communities after 48 h of incubation (n = 3).
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Figure 9. Blue coloration and clearing zones produced by representative bacterial isolates from P. losbanosensis (PL) and E. eugeniae (EE) vermicasts capable of N fixation on nitrogen-free malate media (a), P solubilization on Pikovskaya’s agar (b), and K solubilization on Aleksandrow agar (c) after 5 d of incubation (n = 2). Zones were first observed after 24 h of incubation, with increasing diameters as time progressed.
Figure 9. Blue coloration and clearing zones produced by representative bacterial isolates from P. losbanosensis (PL) and E. eugeniae (EE) vermicasts capable of N fixation on nitrogen-free malate media (a), P solubilization on Pikovskaya’s agar (b), and K solubilization on Aleksandrow agar (c) after 5 d of incubation (n = 2). Zones were first observed after 24 h of incubation, with increasing diameters as time progressed.
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Figure 10. Zone of blue coloration produced by bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) vermicasts capable of N fixation on nitrogen-free malate media after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
Figure 10. Zone of blue coloration produced by bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) vermicasts capable of N fixation on nitrogen-free malate media after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
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Figure 11. P solubilization index (SI) of bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) capable of P solubilization on Pikovskaya’s agar after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
Figure 11. P solubilization index (SI) of bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) capable of P solubilization on Pikovskaya’s agar after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
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Figure 12. K solubilization index (SI) of bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) capable of K solubilization on Aleksandrow agar after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
Figure 12. K solubilization index (SI) of bacteria isolated from P. losbanosensis (PL) and E. eugeniae (EE) capable of K solubilization on Aleksandrow agar after 5 d of incubation (n = 2). Vertical bars represent the standard error of the mean. Means separated by different letters are significantly different (Tukey’s mean separation test, p < 0.05).
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Table 1. The total number of bases, reads, GC (%), AT (%), Q20 (%), and Q30 (%) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by next-generation sequencing using Illumina MiSeq.
Table 1. The total number of bases, reads, GC (%), AT (%), Q20 (%), and Q30 (%) of P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by next-generation sequencing using Illumina MiSeq.
VermicastsTotal Read
Bases (bp)		Total ReadsGC (%)AT (%)Q20 (%)Q30 (%)
P. losbanosensis (PL)98,439,040327,04056.57643.4287.44275.061
E. eugeniae (EE)60,496,184200,98452.81447.1988.73377.007
Table 2. Alpha diversity indices for bacterial communities in the P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME.
Table 2. Alpha diversity indices for bacterial communities in the P. losbanosensis (PL) and E. eugeniae (EE) vermicasts generated by QIIME.
VermicastsQuality ReadsObserved OTUsChao1ShannonInverse SimpsonGood’s Coverage
P. losbanosensis (PL)4734280280.06.68340.98061.0
E. eugeniae (EE)6111133133.254.28160.85810.9997
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MDPI and ACS Style

Mapile, M.R.F.; Aspe, N.M.; Obusan, M.C.M. Vermicast Analysis with the Earthworm Species Pheretima losbanosensis (Crassiclitellata: Megascolecidae): Bacterial Profiles for Potential Applications in Agriculture. Appl. Sci. 2023, 13, 10364. https://doi.org/10.3390/app131810364

AMA Style

Mapile MRF, Aspe NM, Obusan MCM. Vermicast Analysis with the Earthworm Species Pheretima losbanosensis (Crassiclitellata: Megascolecidae): Bacterial Profiles for Potential Applications in Agriculture. Applied Sciences. 2023; 13(18):10364. https://doi.org/10.3390/app131810364

Chicago/Turabian Style

Mapile, Maria Reynalen F., Nonillon M. Aspe, and Marie Christine M. Obusan. 2023. "Vermicast Analysis with the Earthworm Species Pheretima losbanosensis (Crassiclitellata: Megascolecidae): Bacterial Profiles for Potential Applications in Agriculture" Applied Sciences 13, no. 18: 10364. https://doi.org/10.3390/app131810364

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