The Overlooked Decomposers: Effects of Composting Materials and Duration on the Mesofauna Mediating Humification

Abstract

Compost fauna act by releasing various enzymes that break down organic matter into a stable, agriculturally useful products. Mesofauna are the least studied compared to micro- and macrofauna, with the existing studies relying on classical methods such as morphological identification, essentially leaving out cryptic taxa. We sought to evaluate the ecological response of the mesofauna community to different composting materials and durations. Total mesofauna community 18S rRNA was purified in triplicate from lantana-based, tithonia-based, grass-based, and mixed (lantana + tithonia + grass)-based compost heaps after 21, 42, 63, and 84 days of composting and sequenced using the Illumina Miseq platform. Before performing statistical data analysis, we used the Divisive Amplicon Denoising Algorithm version 2 workflow for bioinformatic analyses. The composting duration, but not the composting materials, significantly influenced the total population and composition of the mesofauna communities. The composting materials and duration significantly affected the dispersion and uniqueness of the compost mesofauna communities. Canonical correspondence analysis of the compost’s physical–chemical and biological states showed a significant influence of the materials on the mesofauna community colonization capacity. The mesofauna communities had a significant response to the composting duration. This, therefore, presents them as valuable tools for understanding the temporal evolution of compost.

1. Introduction

Sustainable soil fertility enhancement relies on recycling locally available renewable resources and techniques such as composting, mulching, biomass transfer, and green manuring [1]. Compost is superior to other single input uses [2,3] since it supplies the soil with important nutrients and organisms that support nutrient cycling [4,5]. There are several methods of composting, and thermophilic composting is widely accepted. This is because the technique can sanitize pathogens and weeds in raw manure, which is not achievable through other composting methods [6,7]. The process involves the controlled cycling of organic wastes through the mediation of litter chemistry and decomposition communities to produce organic fertilizers, dissipating temperature [8]. The decomposition communities are categorized as microfauna, mesofauna, and macrofauna, which all uniquely contribute to the composting process through rate-limiting functions [9,10]. However, mesofauna are the least studied decomposition biota, with most existing studies relying on classical methods [11,12,13].

Mesofauna are a small invertebrate group, measuring 0.2 to 2.0 mm, and are ubiquitous in diverse ecosystems. They are the higher organisms based on size and trophic levels in ecosystems, with some belonging to feeding guilds that rely on microfauna [9,14,15]. The group is generally regarded as an intermediate taxonomic group since they are larger than microfauna such as bacteria and protozoa but smaller than macrofauna such as earthworms and Diptera [16]. They participate in ecosystem turnover by fragmenting organic matter, thus accelerating microbial decomposition [17]. Through this, mesofauna communities cycle soil nutrients and facilitate below-ground biodiversity, enhancing soil health. Among the major mesofauna taxa in the ecosystems are mites, pauropods, symphylans, proturans, and springtails, all mediating various roles in their environments [17,18,19].

The mesofauna communities are reciprocally influenced by the prevailing physical, biogeographical, and chemical environmental conditions, impacting their composition and functional capacity [19]. Consequently, ecosystem modifications contribute substantially to deep changes in biogeochemical properties and the mesofauna population and functions. This presents mesofauna as an ideal tool for establishing the maturity and quality of compost due to its direct functional sensitivity and correlation with the environment [13]. Understanding which specific mesofauna species participate in ecosystem processes will contribute to optimizing composting techniques and maximizing their benefits. Moreover, most studies on compost fauna have focused on municipal wastes and manure-only decomposition. This leaves out the farm-yard manure composting that involves Supplemental Materials such as tithonia and dry maize stalks.

Despite prokaryotes and fungi receiving much scientific attention and being regarded as main decomposers, materials such as lignin are difficult for these organisms to break down, requiring larger organisms such as mesofauna [16,19,20]. Mesofauna also play host to microorganisms by harboring them in their body systems, e.g., as the gut microbiome, where they play symbiotic roles [21]. Notwithstanding their ecological importance, a comprehensive and systematic assessment of the community structure and function of the mesofauna is still lacking.

Therefore, we conducted this study to evaluate (i) the ecological response of the mesofauna community to composting substrates and the temporal shifts in their physical–chemical state and (ii) their role in stimulating decomposition using next-generation sequencing.

2. Materials and Methods

2.1. Research Site and Treatments

We set the field experiment at the SysCom project Kenya (Farming Systems Comparison in the Tropics; Kenya) trial site at Thika, Kenya (01°0.231′ S 37°04.747′ E), between September 2020 and December 2020 [22,23], www.system-comparison.fibl.org (accessed on 14 April 2024)).

Our treatment selection was based on the standard composting practices in the tropics (

Table 1. The experiment treatments and respective compost feedstock combination.

Treatment	Main Composting Materials
Lantana-based compost	Fresh cow dung manure, chopped dry maize stalks, chopped lantana twigs (4:2:1 w/w)
Tithonia-based compost	Fresh cow dung manure, chopped dry maize stalks, chopped tithonia twigs (4:2:1 w/w)
Grass-based compost	Fresh cow dung manure, chopped dry maize stalks, chopped grass clippings (4:2:1 w/w)
Lantana + tithonia + grass-based compost [Mixed]	Fresh cow dung manure, chopped dry maize stalks, chopped * (lantana + tithonia + grass) (4:2:1 w/w)

* Equal amounts of grass clippings, tithonia, and lantana twigs were used to prepare the mixed treatment.

). Cattle manure was sourced from a zero-grazing dairy unit close to the trial site, while dry maize stalks and green matter (tithonia, lantana twigs, and grass clippings) were obtained from the site and nearby farms. Each compost treatment was set under a composting shade in three replicates. Layering of the materials was performed as per the standard practice in sub-Saharan Africa [23]. Small dry twigs and pebbles were first laid on a flat surface under the composting shade. This was followed by a layer of chopped dry maize stalks, then a layer of cow dung manure. We finally added a layer of green materials (lantana, tithonia, grass, or a mixture for the specific treatments). At the beginning of composting, the moisture content of each heap was adjusted to approximately 60%. Compost heap aeration was performed by turning it every four days during the first 20 days, then weekly until 84 days of composting.

2.2. Evaluation of the Physical–Chemical Evolution of Compost

Daily temperature monitoring was performed using a compost thermometer (model: WIKA 110824862-EN 13190; Louisville, KY, USA) at three points on the individual compost heaps, as described by Matheri et al. [23]. The pH, moisture, total Kjeldahl nitrogen content, total organic carbon content, and Olsen phosphorous content were analyzed gravimetrically according to the protocols of Okalelebo et al. and Adamtey [24,25].

2.3. Sample Collection and Total Compost DNA Extraction

Compost samples were collected from five points on each pile on the 21st, 42nd, 63rd, and 84th day. We used day 21 of composting as the baseline since the composting materials (maize stovers, cow dung manure, and lantana/grass/tithonia) were not sufficiently homogenized during the earlier days of composting and therefore would have biased the sampling. A top-to-bottom sampling approach was used with sharp pre-sterilized shovels (under 70% ethanol). The samples were then transported on ice to the sample room and stored at −20 °C pending total DNA extraction.

Total compost microbial DNA extraction from the samples was performed at the Kenyatta University plant transformation laboratories in Nairobi, Kenya. We used the PureLink™ Microbiome DNA Purification Kit (Catalog number: A29790, Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions; www.thermofisher.com/ke/en/home/life-science, (accessed on 14 April 2024). DNA quality and concentrations were measured using a 2% agarose gel and a NanoDrop (Maestro-gen) before shipping them on dry ice to the Molecular Research DNA Lab (www.mrdnalab.com, (accessed on 14 April 2024). Shallowater, TX, USA) for sequencing.

2.4. Compost Mesofauna Microbiome Amplification

The 18s PCR primer sets EUK1391F (5′GTACACACCGCCCGTC-3′) and EukBr (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) [26] were used for the amplification of non-fungal eukaryotic DNA which includes mesofauna DNA. Eukaryotic DNA amplification was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD, USA). We set the PCR conditions to be 30 cycles of 95 °C for 30 s, 53 °C for 40 s, and 72 °C for 1 min, with a final elongation step at 72 °C for 10 min. The PCR amplicons were then visually quantified using a 2% agarose gel [23]. Equimolar quantities of the obtained eukaryotic PCR amplicons were multiplexed using unique indices, pooled, and sequenced using Illumina MiSeq next-generation technology at MR DNA (www.mrdnalab.com, (accessed on 14 April 2024) Shallowater, TX, USA). We then trimmed the barcodes and amplicon primer sequences. De-noising and filtering of low-quality sequences with reads <300 base pairs were performed after phred20-based quality trimming, while sequences with ambiguous base calls and homopolymer runs (exceeding 5 bp) were removed [27]. The raw 18S sequences were submitted to the NCBI sequence archive under the accession number PRJNA822850 (https:/www.ncbi.nlm.nih.gov/sra/PRJNA822850 (accessed on 14 April 2024)).

2.5. Bioinformatics and Sequence Data Statistical Analyses

The raw 18S amplicon sequences obtained from the Illumina MiSeq platform were first assessed for sequence quality using FastQC (v 0.11.6). Low-quality reads and primers were then trimmed using cutadapt (v.4.1). Subsequently, the filtered reads underwent preprocessing utilizing the Divisive Amplicon Denoising Algorithm 2 (DADA2) v 1.26.0 [28]. After that, the amplicon sequence variants’ (ASVs’) taxonomic classification was inferred using the pr2 protist pre-trained database [29]. The filtered ASV abundance matrix, taxonomy table, and metadata were merged into a phyloseq object, facilitating the determination of differential mesofauna abundance using the phyloseq (v1.41) package [30].

Mesofauna alpha diversity metrics were computed and tested for normality and significance using the alpha function of the microbiome (v 1.22.0) package. Permutational Multivariate Analysis of Variance (PERMANOVA) and betadisper were used to compare mesofauna population distributions across different compost treatments and composting days. The metagMisc (v.0.04) package aided in the visualization of the phyloseq object, presenting mesofauna relative abundances and percentages across compost treatments and composting days. The beta diversity analysis utilized Principal Coordinate Analysis (PCoA) to elucidate the contribution of compost treatments and composting duration to mesofauna variation. Shared mesofauna communities were determined through Venn diagrams using Euler (v7.0.0). Lastly, we utilized Canonical Correlation Analysis (CCA) following a step-wise model-choosing procedure to identify and measure associations between mesofauna distribution and the abiotic status of the compost [31].

3. Results

We obtained 28,253,246 paired reads spanning the V7–V8 variable regions of eukaryotic 18s rRNA. The DADA2 preprocessing led to 19,109,631 reads and 1159 ASVs for taxonomic assignment. From the analysis, 1158 species of eukaryotes were determined to facilitate the decomposition of organic matter and were influenced by the different sources of green composting materials (Supplementary File S1).

3.1. Alpha Diversity of Mesofauna Community under the Influence of Composting Materials and Duration

We observed significant differences in the mesofauna populations (Chao) between the different sampling time points (Kruskal–Wallis chi-squared = 11.517, p = 0.009236) (Figure 1). However, there were no significant differences in all other alpha diversity metrics among the composting treatments and durations.

3.2. PERMANOVA and Species Composition of Compost Mesofauna

The overall mesofauna community composition was substantially (36.0%) attributable to composting duration (R² = 0.350, F = 1.974, p = 0.0004) but not to differences in dispersion. However, there were no significant differences in dispersion among the composting treatments.

There was preferential colonization of specific mesofauna species on the different composting materials. For instance, Pergamasus canestrinii displayed a higher abundance in the lantana (45.4%) and tithonia (28.6%) compost regimes compared to the grass (2.5%) and mixed (3.5%) regimes. Similarly, Lepidocyrtus paradoxus showed elevated percentages in the mixed (10.0%) and tithonia (25.5%) compost regimes compared to the grass (2.5%) and lantana (2.7%) regimes (Figure 2a, Supplementary File S2). The diversity and dynamics of the mesofauna populations across the sampling time points showed that Cryptopygus antarcticus dominated, representing 31.9% of the total mesofauna population on the 21st day, followed by a significant increase to 69.3% on day 42 and gradually declining to 2.5% by the 84th day. Lepidocyrtus paradoxus showed a minor presence on day 21 (0.2%) but experienced a substantial surge to 46.9% by day 84. Sminthurides aquaticus displayed fluctuations in abundance, peaking at 25.9% on day 42 and gradually declining afterward. Some species, including Aphodanoetus teinophallus, Cosmoglyphus spp., and Culicoides variipennis, exhibited sporadic occurrences with minimal representation across the sampling time points (Figure 2b, Supplementary File S3).

3.3. Distribution and Uniqueness of Compost Mesofauna Communities

The Bray–Curtis dissimilarity-based analysis showed a significant (45.9%) distinction among the composting treatments and time points (p ≤ 0.001). The mesofauna community distinction was highly variable in the lantana-based compost and on the 84th day of composting (Figure 3a,b). The different compost treatments had more shared species and fewer unique ones while the different composting durations bore more unique mesofauna species. Overall, the composting durations had more distinct ASVs compared to the composting treatments. The lantana-based compost had the highest number of unique ASVs among the treatments, while day 21 had the most unique ASVs among the composting durations (Figure 3c,d).

3.4. Influence of Environmental Factors on Compost Mesofauna Community Structure

The ordination biplots showed the compost abiotic characteristics that significantly influenced the mesofauna assemblages (p = 0.001). Temperature and organic carbon content produced the highest rate of change in the mesofauna communities. These physical–chemical elements and pH and total nitrogen, nitrate, and phosphorous contents significantly influenced the mesofauna community structure (Figure 4a). No groupings were observed based on composting materials, while the composting duration plot showed that ammonia, phosphorous, and total nitrogen contents were major community structure shapers after 21 days of composting. On the other hand, pH and nitrate content had a higher influence on the composition and abundance on the 42nd day of composting (Figure 4b).

The direction of the arrows indicates the direction of the maximum change of that variable. The length of the arrow is proportional to the rate of change.

4. Discussion

Our results showed that the composting duration significantly influenced the number of species (Chao1) while the composting treatments (feedstock) did not. The high mesofauna population on day 84 of composting indicates that the compost environment was stable, allowing for mesophilic colonization. These organisms are generally heat-sensitive and have low loads during the thermophilic phase of composting [32]. All composting treatments conferred a similar mesofauna diversity and, therefore, are equally capable of enhancing ecosystem resilience through functional redundancy. The lack of influence of the treatment on the mesofauna species means they responded similarly, irrespective of the materials and duration of composting. Our findings corroborate the results of Barus et al. [33], who averred that adding organic matter has little immediate influence on the mesofauna, indicating that time was a factor in the ecological response of the community.

The top 30 mesofauna species reported in this study have also featured as the top taxa in other studies with diverse roles in nutrient cycling and phyto-protection. These taxa have been consistently shown to be in relatively higher abundance compared to other species, colonizing many different habitats such as grasslands, forest soils, and agricultural farms [34,35,36]. For example, Macrocheles mites are the dominant species in soil ecosystems, where they play a significant predatory role, thus providing an ecological balance for nematodes and dung beetles [37]. Pergamasus canestrinii is a species of mites that dominates forest soils, grazing lands, and decaying manures and is among the top 14 dominant species in many habitats [38,39] and is associated with breaking down organic matter and controlling microbial populations through predation [40].

The species Lepidocyrtus paradoxus, which was predominant in the tithonia-based compost, is a springtail that contributes to leaf litter decomposition through physical fragmentation [41]. They also regulate microbial population in ecosystems such as compost by grazing on fungi and bacteria, ultimately excreting nutrients such as nitrogen and phosphorous. This effectively positions Tithonia diversifolia as a superior compost amendment, encouraging flora that aid in nitrogen and phosphorous cycling. On the other hand, the absence of this species during the thermophilic phase (day 21) was due to the unsuitable high temperatures, limiting their survival [42,43]. The gradual increase beginning on day 42 and peaking on day 84 was commensurate with the gradual decline in pile temperatures, encouraging colonization and grazing of mesophilic micro-organisms which are preferential to the species. Pergamasus canestrinii is a soil mite that is common in leaf litter, contributing to nutrient cycling mainly through indirect decomposition of organic matter by relying on microorganism guilds [44]. The higher population of this species in the lantana-based compost is explained by the relatively high microbial population in this treatment that was observed by Matheri et al. [23]. The dominance of this species at day 21 of composting compared to other species points to its considerable adaptation to high temperatures. Moreover, this period has higher microbial activity, providing food guilds for the species. Pergamasus canestrinii and other mites have been reported to have resilience against high ecosystem temperatures though mechanisms such as the production of heat-stable enzymes, rapid reproduction, and burrowing into cooler pockets of the environment [45,46,47].

The species Cryptopygus antarcticus adapts to fluctuations in ecosystem temperature, moisture, and nutrients [44,48], hence its presence and dominance at all composting time points in our findings. This collembolan is native to undisturbed soils and has been reported among the most abundant mesofauna taxa in diverse ecosystems [49,50,51,52]. This taxon’s thermal and moisture preferences explain its dominance in thermophilic compost environments, out-competing other species [51]. The population increases of Mayetiola destructor beginning on day 42 of composting suggest a potential response to maturation or changes in compost temporal conditions. This makes this species a feasible eco-indicator of compost maturity. Despite being a plant pathogen, this taxon has been reported to contribute to tilts in carbon metabolism and the C:N ratio by decreasing C compounds and increasing N compounds in plants [53].

The substantially higher unique species observed in the lantana-based treatment affirms the differential impact of composting material on the mesofauna community structure. Lantana has been reported to spur the recruitment of specialized decomposers [23] and, therefore, is suitable to mediate a more robust ecological response. Compared to other treatments, lantana is richer in secondary metabolites such as alkaloids and phenolics [54]. The greater number of shared species (ASVs) between the different composting durations compared to the different composting materials suggests a more significant influence of composting duration than the nature of the feedstock. The decomposition process naturally recruits certain organisms regardless of the available organic matter. These taxa are generally opportunistic and indiscriminately feed on organic matter rather than selectively colonizing specific materials [17,55,56].

The abiotic factors explain how composting materials shape the major mesofauna community. We attributed the high influence of total nitrogen and carbon contents on the mesofauna communities to the labile nature of these elements. Some mesofauna species are part of a large nitrogen-cycling community in ecosystems. Therefore, significant changes in nitrogen during the composting process were responsible for their drastic changes [17]. Compost temperature is a principal driver of mesofauna assemblages due to their sensitivity to thermal fluctuations, which impacts their metabolic and reproductive capacities [57].

Additionally, temperature-mediated shifts in microbial activity indirectly influence mesofauna dynamics due to their role as hosts and precursor providers of microorganisms. These temporal shifts in communities are related to carbon levels, which are highly correlated with compost temperature [23,55,56,58]. Previous studies have also identified phosphorus as a rate-limiting element during litter decomposition, with a strong association with fauna [59]. This corroborates previous studies that showed that mesofauna are suitable predictors for ecosystem functioning, where the decomposition status was correlated with the properties of the materials [60,61].

5. Conclusions

The significant differences among the different composting time points uncover the dynamic response of mesofauna communities to temporal variations in organic matter during decomposition. This effectively positions the mesofauna community as a superior tool for fingerprinting different phases of compost succession over time. However, the composting treatments had less influence on the mesofauna communities compared to the composting duration. Our study points out the functional redundancy of composting substrates on the mesofauna community structure since they exhibit similar ecological responses to different materials.