PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0286320 PONE-D-22-30019 Research Article Biology and life sciencesOrganismsEukaryota Biology and life sciencesMycologyFungal structure Biology and life sciencesEcologyCommunity ecologyCommunity structure Ecology and environmental sciencesEcologyCommunity ecologyCommunity structure Biology and life sciencesEcologyBiodiversity Ecology and environmental sciencesEcologyBiodiversity Biology and life sciencesEcologyEcosystems Ecology and environmental sciencesEcologyEcosystems Biology and life sciencesEcologyEcological metricsSpecies diversity Ecology and environmental sciencesEcologyEcological metricsSpecies diversity Biology and life sciencesMolecular biologyMolecular biology techniquesArtificial gene amplification and extensionPolymerase chain reaction Research and analysis methodsMolecular biology techniquesArtificial gene amplification and extensionPolymerase chain reaction Physical sciencesChemistryChemical compoundsNitrates Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks Diverse green materials profoundly influence eukaryotic community structure https://orcid.org/0000-0002-8729-8529 Matheri Felix Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Validation Visualization Writing – original draft Writing – review & editing 1 2 * Kambura Anne Kelly Conceptualization Data curation Formal analysis Investigation Methodology Supervision Validation Visualization Writing – original draft Writing – review & editing 3 Mwangi Maina Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Writing – review & editing 1 Karanja Edward Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Resources Validation Writing – original draft Writing – review & editing 2 Adamtey Noah Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Writing – original draft Writing – review & editing 4 Wanjau Kennedy Data curation Formal analysis Software Validation Visualization Writing – review & editing 5 Mwangi Edwin Conceptualization Formal analysis Investigation Methodology Validation Writing – original draft Writing – review & editing 2 Tanga Chrysantus Mbi Funding acquisition Investigation Methodology Project administration Resources Software Validation Visualization Writing – original draft Writing – review & editing 2 https://orcid.org/0000-0002-5498-3298 Bautze David Funding acquisition Investigation Resources Software Visualization Writing – review & editing 4 Runo Steven Conceptualization Formal analysis Funding acquisition Investigation Methodology Project administration Resources Supervision Validation Writing – review & editing 1 Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University (KU), Nairobi, Kenya International Centre for Insect Physiology and Ecology (icipe), Nairobi, Kenya Department of Agricultural Sciences, Taita Taveta University (TTU), Voi, Kenya Research Institute of Organic Agriculture (FiBL), Frick, Switzerland International Livestock Research Institute (ILRI), Department Animal and Human Health, Nairobi, Kenya Bhattacharjya Sudeshna Editor ICAR-Indian Institute of Soil Science, INDIA

The authors have declared that no competing interests exist.

 * E-mail: fmatheri@icipe.org 31 5 2023 2023 18 5 e0286320 31 10 2022 15 5 2023 2023 Matheri et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Thermophilic composting is a promising soil and waste management approach involving diverse micro and macro-organisms, including eukaryotes. Due to sub-optimal amounts of nutrients in manure, supplemental feedstock materials such as Lantana camara, and Tithonia diversifolia twigs are used in composting. These materials have, however, been reported to have antimicrobial activity in in-vitro experiments. Furthermore, the phytochemical analysis has shown differences in their complexities, thus possibly requiring various periods to break down. Therefore, it is necessary to understand these materials’ influence on the biological and physical-chemical stability of compost. Most compost microbiome studies have been bacterial-centric, leaving out eukaryotes despite their critical role in the environment. Here, the influence of different green feedstock on the fungal and non-fungal eukaryotic community structure in a thermophilic compost environment was examined. Total community fungal and non-fungal eukaryotic DNA was recovered from triplicate compost samples of four experimental regimes. Sequencing for fungal ITS and non-fungal eukaryotes; 18S rDNA was done under the Illumina Miseq platform, and bioinformatics analysis was done using Divisive Amplicon Denoising Algorithm version 2 workflow in R version 4.1. Samples of mixed compost and composting day 84 recorded significantly (P<0.05) higher overall fungal populations, while Lantana-based compost and composting day 84 revealed the highest fungal community diversity. Non-fungal eukaryotic richness was significantly (P< 0.05) more abundant in Tithonia-based compost and composting day 21. The most diverse non-fungal eukaryotic biome was in the Tithonia-based compost and composting day 84. Sordariomycetes and Holozoa were the most contributors to the fungal and non-fungal community interactions in the compost environment, respectively. The findings of this study unravel the inherent influence of diverse composting materials and days on the eukaryotic community structure and compost’s biological and chemical stability.

 http://dx.doi.org/10.13039/501100015593 Biovision Foundation for Ecological Development 1040 Coop Sustainability Fund 1040 Liechtenstein Development Service (LED) 1040 Swiss Agency for Development and Cooperation (SDC) 1040 DAAD-Africa 91712524 https://orcid.org/0000-0002-8729-8529 Matheri Felix United Kingdom’s Foreign, Commonwealth and Development Office (FCDO) Swedish International Development Cooperation Agency (Sida) Swiss Agency for Development and Cooperation (SDC) Federal Democratic Republic of Ethiopia Government of the Republic of Kenya This research received partial financial support from Biovision Foundation (Grant No: 1040), Coop Sustainability Fund (Grant No: 1040), Liechtenstein Development Service (LED) (Grant No: 1040) and Swiss Agency for Development and Cooperation (SDC) (Grant No: 1040),. The authors also acknowledge partial financial support from the DAAD-Africa through the "In-Country/ In-Region Scholarship Programmes Eastern Africa" (Grant No: 91712524) We also gratefully acknowledge the support of icipe core funding provided by United Kingdom’s Foreign, Commonwealth and Development Office (FCDO); the Swedish International Development Cooperation Agency (Sida); the Swiss Agency for Development and Cooperation (SDC); the Federal Democratic Republic of Ethiopia; and the Government of the Republic of Kenya.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”. Data Availability The raw ITS and 18S sequences were submitted to the NCBI sequence archive with accession number PRJNA822850 (https://www.ncbi.nlm.nih.gov/sra/PRJNA822850). Introduction

Thermophilic composting, also known as hot rotting, is the degradation of organic material driven by different categories of organisms that bring about turns of high and low temperatures [1, 2]. This composting method is ideal for managing organic wastes from systems such as agricultural, municipal, and food industries that would otherwise be hazardous to the environment [3]. Microorganisms involved in composting include prokaryotes, protozoa, fungi, and other eukaryotes, with prokaryotes being dominant and most studied [4, 5]. Though less studied, eukaryotes are an important microbial category in the ecosystem with diverse feeding and genetic guilds [6, 7]. Eukaryotes degrade recalcitrant material such as Carbon-rich polymers, thus contributing to nutrient cycling in the compost environment [8]. The complex structure and function of a large number of compost microorganisms are directly affected by various environmental factors such as temperature, moisture, Carbon/Nitrogen ratio, and pH, among others [9]. Consequently, compost eukaryotes represent a largely unexplored frontier in microbial ecology and hold inordinate potential for discovering new communities and functional assemblages.

In agricultural systems, cattle manure is the primary composting material due to its ready availability and its harboring of important nutrients and microbes for plant growth [10]. However, due to suboptimal levels of important nutrients in cattle manure during composting, the common practice requires supplemental feedstock material. Green materials such as fresh grass clippings and fresh twigs of Lantana camara and Tithonia diversifolia have been recommended and used to supplement nitrogen levels in soils and compost [11, 12]. Some of these materials have been reported under invitro studies as having inhibitory properties on fungal and non-fungal organisms [13, 14]. The phytochemical complexities of Lantana camara and Tithonia diversifolia have been previously studied. Lantana camara has been reported to contain more complex polymers and thus possibly requires more microbial categories to break down into agriculturally useful material. For example, the phytochemical composition analysis of L. camara showed 23.3% crude fiber, 26% cellulose, 16.2% lignin, and 21% hemicellulose, while T. diversifolia contains 11.2%, 17%, 7%, and 16% of these elements respectively [15–17]. Furthermore, the different complexities of the composting material could require assorted composting times to produce a biogeochemically stable product. It is, therefore, necessary to evaluate the influence of composting time on the biological and physical-chemical quality of compost.

Numerous studies have assessed the prokaryotic community in the composting process using culture-dependent and culture-independent methods [2, 4, 6, 10]. However, there is still a limited understanding of eukaryotic communities’ structure in the composting process, concerning the composition and complexity of the composting materials especially utilizing high-throughput sequencing technology. The effect of the Lantana camara and Tithonia diversifolia on microbial community structure in complex ecosystems such as the compost environment has not also been done, despite their widespread adoption by farmers as soil nutrient amendment materials.

This study comprehensively assessed fungal and non-fungal eukaryotic communities associated with the co-composting of assorted nitrogenous green material and composting time. The study hypothesized that different composting materials and days influenced eukaryotic communities differently. The culture-independent, high-throughput sequencing Illumina MiSeq platform was used for library preparation of the fungal and non-fungal eukaryotic communities in the different compost environments. Furthermore, the study evaluated the correlation and co-dependence of compost physical-chemical factors and their influence on fungal and non-fungal eukaryotic communities.

 Materials and methods Compost heaping and sampling

The composting materials that were common to all treatments were sourced from the same farm around the composting site. Particularly, raw manure for all the compost treatments was obtained from a single dairy unit. Compost preparation and heaping were done on the same day in the Long-Term Farming Systems Comparison trial site at Thika, Kenya (01 ° 0.231’ S 37 ° 04.747’ E) [www.system-comparison.fibl.org, 18]. The site was established by the Research Institute of Organic Agriculture (FiBL) and local partners, including the International Centre for Insect Physiology and Ecology (ICIPE) and Kenya Agricultural and Livestock Research Organization (KALRO).

Treatments were based on common farmer practices in Kenya and available sources of nitrogenous material for composting in the region [18, 19]. All compost treatments had an equal ratio of 4:2:1 w/w for fresh cow dung manure, dry maize stalks, and nitrogenous green materials, respectively. The different sources of nitrogenous green material treatments were as per Table 1.

10.1371/journal.pone.0286320.t001 Compost feedstock and green material types informing the treatments used for the experiment.
Materials used Treatment/label
Fresh cow dung + dry maize stalks + fresh Lantana camara twigs Lantana-based compost (L)
Fresh cow dung + dry maize stalks + fresh Tithonia diversifolia twigs Tithonia-based compost (T)
Fresh cow dung + dry maize stalks + fresh grass clippings Grass-based compost (G)
Fresh cow dung + dry maize stalks + fresh grass clippings, fresh Tithonia diversifolia, and Lantana camara twigs in the ratio of 1:1:1 Lantana, Tithonia, Grass (Mixed)-based compost (LTG)

*Wood ash (1kg) and soil (5kg) were sprinkled after every layer was heaped.

Composting materials described above were individually cut into small pieces (3–5cm long) to enable uniform and faster breakdown. Compost heaping was done in triplicates for each of the four compost treatments. Each heap was done by laying small dry twigs on a flat leveled composting surface, followed by a layer of dry chopped maize stalks. Cow dung manure from zero-grazed cattle was used for the next layer, and, finally a layer of green material (Lantana/Tithonia/Grass/ mixture of Lantana, Tithonia, and Grass). The heaping process was repeated four times for each compost pile and heap moisture content was adjusted to about 60%, at the beginning of composting as recommended by [20]. Compost heaps aeration was done by turning every four days during the first 20 days and weekly for the following days till 84 days of composting. Sampling was done every 21 days, with the first 21 days of composting serving as the baseline. Therefore, sampling was done at 21, 42, 63, and 84 composting days.

 Physical-chemical characterization of compost treatments

The daily temperature was monitored using a compost thermometer (model: WIKA 110824862-EN 13190; Louisville, USA) at three locations on the heap by inserting the thermometer halfway between the top and bottom of the heap to the maximum probe depth (45 cm). Sampling for other physical-chemical parameters was done on the sampling days described above. The pH of the compost (1:10 w/v waste: water extract), moisture, germination index, and Carbon dioxide emission during sampling days (mg CO2 g-1d-1) were done as described by [21]. Total Kjeldahl nitrogen (TKN) was analyzed using the Kjeldahl method. The total organic Carbon and total phosphorus (TP) (Olsen P) were analyzed according to [22].

 Sampling for ITS and 18S rDNA analysis of compost eukaryotic communities

Compost samples for total DNA extraction were collected on days 21, 42, 63, and 84 of the composting process. Two set-sampling was done on each triplicate heap of each treatment from five different positions using a sharp shovel that was, pre-cleaned with 70% ethanol as described by [20, 23]. The samples were transported to ICIPE and stored at -20°C. Total DNA extraction of the samples was carried out at the Kenyatta university plant transformation laboratories, Nairobi, Kenya.

 Compost microbiome total DNA extraction and amplification

Total compost DNA was extracted from triplicate compost subsamples of each treatment for fungal and other eukaryotic communities DNA extraction. The PureLink™ Microbiome DNA Purification Kit (Catalog number: A29790) as per the manufacturer’s instructions (www.thermofisher.com/ke/en/home/life-science). Each extracted sample’s quality and concentrations were measured under 2% agarose gel and NanoDrop (Maestrogen). The purified compost DNA was shipped under dry ice to the Molecular Research DNA Lab (www.mrdnalab.com, Shallowater, TX, USA) for sequencing under the illumine miseq platform.

The PCR primer sets used were ITS1-F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’) for fungal ITS amplification while EUK1391F(5’GTACACACCGCCCGTC-3’) and EukBr (5’-TGATCCTTCTGCAGGTTCACCTAC-3’) for other eukaryotes. Amplification was done in 30 cycles PCR (5 cycles used on PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). The PCR conditions were 95°C for 5 minutes, followed by 30cycles of 95°C for 30 seconds, 53°C for 40 seconds, and 72°C for 1 minute, with a final elongation step at 72°C for 10 minutes performed. Resultant PCR amplicons were visually quantified under 2% agarose gel. There were no fungal amplicons associated with grass-based compost sampled at 42 days. Equimolar quantities of PCR amplicons obtained from the remaining 31 samples (16 non-fungal eukaryotic amplicons from individual composts and 15 fungal amplicons) were multiplexed using unique indices, pooled, and sequenced using Illumina MiSeq next-generation technology at MR DNA (www.mrdnalab.com, Shallowater, TX, USA). Barcodes and amplicon primer sequences were trimmed after sequencing. Low-quality sequences were denoised and filtered out with reads <300 base pairs after phred20-based quality trimming. Sequences with ambiguous base calls and those with homopolymer runs exceeding 5bp were removed [24]. The raw ITS and 18S sequences were submitted to the NCBI sequence archive with accession number PRJNA822850 (https://www.ncbi.nlm.nih.gov/sra/PRJNA822850).

 Bioinformatics analysis

The Divisive Amplicon Denoising Algorithm 2 (DADA2) version 1.20.0 analyzed in R (version 4.1) as described by [25] was used as the primary workflow for analysis. Here, filtering of the reads, learning error rates, dereplication, merging of forward and reverse reads, chimera removal, and taxonomic assignment were done separately for the fungal (ITS) and eukaryotic (18S) sequences. The Unite reference database was used for ITS while, Silva 128 and 132 databases were used for non-fungal eukaryotes (18S) referencing. The products of the workflows were ITS and 18S Amplicon sequence variant (ASV) tables for the two amplicon sets [26].

 Statistical analysis

Data analysis was done using different packages in R version 4.1.2. The data for the various physical-chemical variables were individually subjected to a normality test using the Shapiro test before means separation using ANOVA under the agricolae package (version 1.3–5). Comparing the compost treatments was done per composting day for each physical-chemical parameter, followed by a Tukey posthoc. The distributions of soil physicochemical variables across different compost treatments and composting days were calculated on log-standardized data using the “decostand” function in the Vegan version 2.6–2 package. The resulting distance matrix between samples was plotted in a PCA graph, with the projected direction and magnitude of the distribution for each variable plotted in a separate loading plot. This was followed by the computation of the Pearson correlation matrix using the function “corrplot” in the Corrplot package (version 0.92).

Alpha diversity metrics were calculated using the “estimate_richness” function in phyloseq Observed index was estimated to reflect the number of ASVs in each compost sample and values that have a positive correlation with the species richness. Shannon and Simpson Inverse-Simpson indices of the correlation of diverse species abundance in a sample [27] were also computed. Shapiro and significance tests were calculated before plotting the alpha diversity metrics. The taxonomy table and abundance table were merged with the abundance table, and bar plots of compost treatments and sampling day relative abundance were plotted using the “plot_bar” function. Venn diagrams to show the shared community ASVs among composting treatments and composting days were plotted using the eulerr (version 6.1.1) package. Beta diversity was computed using the Bray-Curtis index to further explore the influence of different composting treatments and composting days and differences on microbial community profiles. The resulting scores were used for PCoA plotting and compared using the PERMANOVA test of significance. A stepwise modeled Canonical Correspondence Analysis (CCA) was done in a Vegan package (version 2.6–2) to show the effect of explanatory physical-chemical variables on the different compost microbiomes A co-expression network detailing the interaction of abundant composting microbiomes was constructed based on microbial abundance using the “plot_net” function in the ggplot2 package (version 3.3.5).

 Results Different compost treatments exhibit distinct physical-chemical properties

Samples from the different compost treatments and composting days were shown to be significantly different (p-value ≤0.05) in terms of most physicochemical properties of treatments in most composting days. There were however notable similarities in the treatments, especially at the latter composting period (Table 2).

10.1371/journal.pone.0286320.t002 Physical-chemical characteristics of different compost treatments on various composting days.
Component Time G L LTG T Trt
Organic Carbon (%) 21 22.73c 28.00a 26.40b 25.77b ***
42 17.67a 17.17ab 16.07bc 15.33c **
63 16.40a 15.57ab 15.20ab 14.47b *
84 13.75a 13.67a 12.87c 13.27b ***
Potassium (%) 21 1.42b 1.52b 1.58ab 1.78a **
42 1.26a 1.16b 1.19ab 1.21ab *
63 1.07a 1.10a 1.10a 1.09a ns
84 1.09a 1.04b 1.01b 1.09a ***
Total Nitrogen (%) 21 0.77b 0.96a 0.82ab 0.97ab *
42 0.75a 0.68ab 0.67ab 0.59b *
63 0.62a 0.58a 0.59a 0.49a ns
84 0.55a 0.60a 0.54a 0.55a ns
Total Phosphorous (%) 21 0.18b 0.21ab 0.24a 0.22ab *
42 0.18a 0.14b 0.13b 0.14b ***
63 0.16a 0.15a 0.15a 0.15a ns
84 0.17a 0.16b 0.14c 0.16ab ***
Moisture Content (%) 21 72.2a 73.9a 70.9a 72.3a ns
42 46.7b 51.1a 44.9c 51.6a ***
63 17.1a 18.8a 19.6a 18.8a ns
84 18.3a 19.3a 19.3a 19.8a ns
Temperature (°C) 21 41.3a 39.7a 38.3a 38.3a ns
42 24.3ab 23.0b 26.3a 25.0ab **
63 25.0a 25.0a 25.0a 27.0a ns
84 22.7a 22.7a 23.0a 22.3a ns
pH 21 8.41a 8.50a 8.48a 8.48a ns
42 8.95ab 9.00a 8.90b 8.99a **
63 8.37a 8.36a 8.25a 8.47a ns
84 8.72a 8.62c 8.55d 8.67b ***

Values with the same superscripts were not significantly different, ns-not significant

* P ≤ 0.05

** P ≤ 0.01 and

*** P ≤ 0.001.

Physical-chemical nature of compost samples revealed significantly distinct compost properties for different composting materials and days (p-value < 0.05). The first principal component (PC1) contributed to 41.7% of the total variance while, the second principal component (PC2), contributed 22.1%. Together, the first two principal components accounted for 69.8% of the total physical-chemical variation of different compost treatments. Notably, day 21 of composting was highly distinct from other composting days. The most variability within groups was recorded in the Tithonia-based compost and composting day 42; Fig 1A and 1B.

10.1371/journal.pone.0286320.g001 <p><bold>A, B:</bold> Principal component analysis (PCA) biplots of compost treatment (A) and composting days (B) according to their physical-chemical properties. Samples were clustered as per different compost types and composting days.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g001" xlink:type="simple"/> </fig> <p>On the other hand, most compost physical-chemical properties were correlated. The compost temperature was positively correlated with Carbon, Phosphorous, and Nitrogen but negatively correlated with pH and Nitrates (<xref ref-type="fig" rid="pone.0286320.g002">Fig 2</xref>).</p> <fig id="pone.0286320.g002" position="float"> <object-id pub-id-type="doi">10.1371/journal.pone.0286320.g002</object-id> <label>Fig 2</label> <caption> <title>Pearson correlation matrix between different physical-chemical variables (C); positive and negative correlations are displayed in blue and red, respectively. Different composting materials and days exhibit distinct biodiversity

Mixed compost (LTG) and composting day 84 recorded significantly (P<0.05) higher overall fungal populations (“Observed”) among the compost types and composting days. Lantana-based compost and composting day 84 had the highest fungal community diversity (Shannon, Simpson, and InvSimpson) (Fig 3A and 3B).

10.1371/journal.pone.0286320.g003 <p>Alpha diversity metrics (Observed, Shannon, InvSimpson, and Simpson) of fungal eukaryotic communities under different composting treatments (A) and composting days. L, T, G, and LTG represent Lantana, Tithonia, Grass, and mixed (Lantana + Tithonia + Grass) based composts respectively. Day 21, day 42, day 63, and day 84 represent the effect of combined compost treatments at 21, 42, 63, and 84 days of composting.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g003" xlink:type="simple"/> </fig> <p>Non-fungal eukaryotic richness was significantly (P< 0.05) more abundant in tithonia-based compost (T) and composting day 21. The most diverse non-fungal eukaryotic biome was in the Tithonia-based compost and composting day 84 (<xref ref-type="fig" rid="pone.0286320.g004">Fig 4A and 4B</xref>).</p> <fig id="pone.0286320.g004" position="float"> <object-id pub-id-type="doi">10.1371/journal.pone.0286320.g004</object-id> <label>Fig 4</label> <caption> <title/> <p>Alpha diversity metrics (Observed, Shannon, InvSimpson, and Simpson) of non-fungal eukaryotic communities under different composting treatments (A) and composting days (B). L, T, G, and LTG represent Lantana, Tithonia, Grass, and mixed (Lantana + Tithonia + Grass) based composts respectively. Day 21, day 42, day 63, and day 84 represent the effect of combined compost treatments at 21, 42, 63, and 84 days of composting.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g004" xlink:type="simple"/> </fig> </sec> <sec id="sec012"> <title>Different composting treatments and days influence the beta diversity of fungal communities distinctly

Overall, the most abundant fungal classes in the compost environment were Sordariomycetes (61% mean relative abundance), Agaricomycetes (8% mean relative abundance), Dothidiomycetes (20% mean relative abundance), Eurotiomycetes (4% mean relative abundance) and Saccharomycetes (2% mean relative abundance); Table 3. The fungal class Sordariomycetes was the most abundant taxa within the compost treatments, particularly in Tithonia-based composts which had a mean relative abundance of 68%; Fig 5A, and Table 3. Sordariomycetes was still the dominant fungal class on all the composting days with the highest abundance recorded at composting day 42 (74%). The class Agaricomycetes was most abundant at composting day 21 compared to other composting days, recording (Fig 5C and Table 3).

10.1371/journal.pone.0286320.g005 <p><bold>A-D:</bold> Relative abundances of fungal and non-fungal eukaryotic classes in various composting treatments (A, C respectively) and sampling days (B and D respectively). L, T, G, and LTG represent lantana, tithonia, grass, and mixed (lantana + tithonia + grass) based composts, respectively. 21, 42, 63, and 84 represent the effect of combined compost treatments at 21, 42, 63, and 84 days.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g005" xlink:type="simple"/> </fig> <table-wrap id="pone.0286320.t003" position="float"> <object-id pub-id-type="doi">10.1371/journal.pone.0286320.t003</object-id> <label>Table 3</label> <caption><title>Mean relative abundance (%) of the most abundant fungal eukaryotic classes in different compost treatments and composting days.
Overall relative abundance Compost treatments Composting days
Class Mean Standard deviation G L LTG T 21 42 63 84
Sordariomycetes 61 18.3 64 56 57 68 67 74 47 60
Dothideomycetes 20 18.8 19 21 20 20 6 12 33 28
Agaricomycetes 8 7.4 9 8 9 6 18 4 6 3
Eurotiomycetes 4 2.4 3 4 7 3 4 4 5 4
Mortierellomycetes 2 3.3 0 3 3 1 0 3 4 1
Saccharomycetes 2 1.6 3 2 2 1 1 1 3 2
Tremellomycetes 1 3.1 0 4 1 0 3 0 0 1
Pezizomycetes 1 0.8 0 1 1 0 1 1 0 1
Cystobasidiomycetes 0 0.8 1 0 0 0 0 0 1 0
Orbiliomycetes 0 0.8 1 1 0 0 0 1 0 0
Microbotryomycetes 0 0.3 0 0 0 0 0 0 0 0
Leotiomycetes 0 0.2 0 0 0 0 0 0 0 0
Rhizophlyctidomycetes 0 0.2 0 0 0 0 0 0 0 0
Laboulbeniomycetes 0 0.1 0 0 0 0 0 0 0 0
Ustilaginomycetes 0 0 0 0 0 0 0 0 0 0
Total 100 100 100 100 100 100 100 100 100

The most abundant non-fungal eukaryotic taxa in all the compost environments were Alveolata (4%), Chloroplastida (13%), Holozoa (58%), Rhizaria (13%), Stramenopiles (8%) and Tublinea (2%); Fig 5B, 5D and Table 4. Non-fungal eukaryotic class, Holozoa was the most abundant taxa with the highest mean relative abundance recorded in Lantana-based compost (77%); Fig 5B and Table 4. Class Holozoa was the most abundant non-fungal eukaryotic taxa across the composting days, with the highest values recorded on composting day 63 (mean relative abundance of 66%); Fig 5D and Table 4.

10.1371/journal.pone.0286320.t004 Mean relative abundance (%) of the most abundant non-fungal eukaryotic classes in different compost treatments and composting days.
Overall relative abundance Compost treatments Days of composting
Class Mean Standard deviation G L LTG T 21 42 63 84
Holozoa 58 23.1 33 77 70 53.5 55 56 66 55
Rhizaria 13 11.8 16 9 15 13.5 28 6 8 12
Chloroplastida 13 24.5 33 1 6 14 4 30 7 13
Stramenopiles 8 4.8 7.8 7.5 6 10 7 6 6 12
Alveolata 4 6 8.5 2 1 3.5 2 1 9 4
Discosea 2 1.3 1 2 1.5 2.5 2 1 3 2
Tubulinea 2 1.4 2 1.5 1 3 3 1 2 2
Total 100 100 100 100 100 100 100 100 100
Principal coordinate analysis (PCoA)

Bray-Curtis beta diversity of samples from different compost types and days showed distinct groupings (Fig 6A–6D). Notably, the percentage of variation in fungal community structure attributed to compost type and composting days is relatively small, with about 73% of the unexplained variance (Fig 6A, and 6C). The widest beta diversities for the fungal and non-fungal communities as influenced by compost treatments were observed in Tithonia-based and Grass-based composts, respectively. The least overlap and variation of diversity among the composting days in both fungal and non-fungal eukaryotic communities were observed on day 21 of composting, Fig 6C and 6D.

10.1371/journal.pone.0286320.g006 <p><bold>A-D:</bold> Principal coordinate analysis (PCoA) ordination plots based on the Bray-Curtis index at 95% confidence. The different compost groups are highlighted by ellipses.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g006" xlink:type="simple"/> </fig> </sec> <sec id="sec014"> <title>Unique fungal and non-fungal eukaryotic taxa within compost environment

Venn diagrams to show the common OTUs and exclusive OTUs among different compost treatments and composting days were generated. Grass-based compost and mixed compost recorded the most unique core fungal taxa among the compost types, with three unique taxa for each of the two treatments (Fig 7A). Lantana-based compost had the highest non-fungal eukaryotic core taxa, recording seven unique taxa (Fig 7B). On the other hand, composting day 21 recorded the highest unique core fungal taxa among all the composting days, with 12 exclusive taxa (Fig 7C) while composting day 21 had the most unique non-fungal taxa (Fig 7D).

10.1371/journal.pone.0286320.g007 <p><bold>A-D:</bold> Venn diagram based on shared major core taxa of fungal and non-fungal eukaryotic communities under compost treatment (A and B), composting days (C and D).</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g007" xlink:type="simple"/> </fig> </sec> <sec id="sec015"> <title>Fungal and non-fungal community interactions within the compost environment

Five (5) fungal classes (Agaricomycete, Eurotiomycetes, Dothideomycets, Saccharomycetes, and Sordariomycetes) were displayed to correlate with each other at different intensities (correlation values between 0.0 and 0.6) as shown in the interaction network. The class Sordariomycetes was the hub taxon contributing to the most network nodes, with 10 taxa belonging to the class. Consequently, this taxon had the most interactions with other fungal classes in the compost environment (Fig 8).

10.1371/journal.pone.0286320.g008 Correlation network showing interactions among fungal eukaryotic classes driving composting.

The network nodes represent genera, whereas the edges represent microbe-microbe interaction weights. Networks were constructed based on the top 20 classes. Various color codes in the networks represent labeled genera within the classes listed on the grid.

On the other hand, seven (7) non-fungal classes (Alveolata, Chloroplastida, Discosea, Holozoa, Rhizaria, Stramenopiles, and Tublinea) were shown to bear the most interactions within the compost environment. Class Holozoa had the most interactions with non-fungal eukaryotic taxa in the compost ecosystem (Fig 9). Taxa under this class supported most close interactions (mainly at a correlation of 0.2) within the non-fungal community; Fig 9.

10.1371/journal.pone.0286320.g009 Correlation network showing interactions among non-fungal eukaryotic classes driving composting.

The network nodes represent genera, whereas the edges represent microbe-microbe interaction weights. Networks were constructed based on the top 20 classes. Various color codes in the networks represent labeled genera within the classes listed on the grid.

 Physical-chemical drivers of compost fungal and non-fungal eukaryotic communities

Canonical correspondence analysis (CCA) ordination plots of environmental factors showed the significant (adj. p-value<0.01) influence of these factors on fungal and non-fungal eukaryotic biomes of compost (Fig 10A and 10B). Most fungal classes were responsive to less ammonia, with Dothideomycetes and Laboulbeniomycetes having the and responded positively to a decrease in other physical-chemical states of compost. The fungal class Agaricomycetes was the most sensitive to Nitrates and Carbon. Non-fungal eukaryotic class Tublinea was uniquely responsive to decreasing temperature, Carbon, and Phosphorous, and increasing Nitrates. The frequency of class Discosea is associated with high pH, total nitrogen, and low nitrate levels.

10.1371/journal.pone.0286320.g010 <p><bold>A, B:</bold> Canonical correspondence analysis (CCA) plots showing the effect of explanatory physical-chemical variables on the different fungal (A) and non-fungal (B) microbiomes using a significance threshold of 0.05. C- Organic Carbon, N- Total Nitrogen, K- Potassium, NO3- Nitrates, NH4- Ammonia.</p> </caption> <graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0286320.g010" xlink:type="simple"/> </fig> </sec> </sec> <sec id="sec017" sec-type="conclusions"> <title>Discussion

Studying eukaryotic communities’ structure in thermophilic composting is crucial to understanding the categories and succession of fungi that are useful for improved soil health and not harmful to humans, plants, and environmental health [28]. This knowledge is also worthwhile in the optimization of compost quality standards for safer crop production [29].

Physical-chemical conditions are indicators of the humification rate that ultimately brings biological and nutritional stability to compost [30]. Humification is preceded by rapid biogeochemical phases which are microbially driven, breaking down complex polymers into organic acids [31]. The high variability within Tithonia-based compost can be attributed to the significant physical-chemical changes within this treatment compared to other treatments along the composting process. The strong positive correlation between factors such as Carbon and temperature points to the co-dependence of these two elements in the physical-chemical nature of compost. The breakdown of Carbon by microbes leads to a temperature increase in the compost environment [1]. This temperature increase is responsible for nutrient mineralization and, ultimately humification of compost [30].

The high fungal biodiversity (richness) at 84 days of composting compared to preceding composting days implies that more extended composting periods are necessary for compost stability [32]. Several authors have reported the recruitment of more fungal categories responsible for the maturation and, ultimately, humification of compost [33–35]. The diversity of the fungal community in Lantana-based compost is attributable to the complexity of Lantana compared to other materials and hence ecosystem recruitment of diverse fungal categories with the capacity to metabolize this material.

The ubiquitous fungi taxa in all composting treatments and days (Sordariomycetes, Agaricomycetes, Dothidiomycetes, Eurotiomycetes, and Saccharomycetes) were reported as resident classes in compost [36–38]. The dominance of class Sordariomycetes in the compost environment indicates its critical role in the evolution of compost and persistence in the compost ecosystem. This class has been reported as having a superior metabolic capability to other fungal classes [39–41].

Classes Alveolata, Chloroplastida, Discosea, Holozoa, Rhizaria, Stramenopiles, and Tublinea have been reported as resident non-fungal eukaryotes that colonize organic wastes [42]. These eukaryotes have been reported as active soil protists [43] with metabolic functions that are essential nutrient cycling pathways. The presence of non-fungal eukaryotic class Chloroplastida in all compost treatments and days affirms the suitability of compost as a soil health input bringing in novel microbes with the ability to improve soil physical-chemical state. Algal biota such as Chloroplastida has been reported as having broad metabolic capabilities in fixing nitrogen, acting as plant growth promotion and disease control [44].

Fungal class Agaricomycetes has been significantly associated with Carbon and Nitrogen cycling in the ecosystem [10] hence their dominant association with nitrates and Carbon during composting. This primes this class as potential soil Carbon and nitrogen cycling microbial category as a suitable candidate for soil improvement inoculants.

The change of unique fungal ASVs in the different composting days implies a clear evolution of fungal communities as influenced by changing nature of materials in compost as well as physical-chemical parameters and vice-versa [28, 36, 45]. The high unique core taxa in Lantana-based compost points to the role of the eukaryotes in the unique degradation of material in this composting treatment. Lantana has been reported as inhibitory to some classes of microorganisms [46–48], therefore requires specialized classes of microbes to break down.

Hub taxa have a direct inhibitory or facilitative role in the proliferation and survival of other microbes affecting overall interconnected communities [49, 50]. The genera in the class Sordariomycetes which is the major fungal hub taxon has been reported as the first line of colonizers of recalcitrant material in soil, therefore breaking this material into simpler forms available for other fungal and prokaryotic communities [39, 41]. The degradation of complex material such as lignocellulose in ecosystems like compost is driven by a synergistic action of oxidative and hydrolytic enzymes that break the linkages within the material [51]. This process requires a variety of interactions among different categories of microorganisms [52].

 Conclusion

This study demonstrates how various sources of green material and composting days affect the evolution of diverse fungal and non-fungal eukaryotic communities. Fungal and non-fungal eukaryotic diversity and abundance changed significantly with compost type and composting days. High throughput ITS and 18S gene sequencing indicated that the dominant classes during composting included Sordariomycetes, Agaricomycetes, Dothidiomycetes, Eurotiomycetes, and Saccharomycetes of fungi. On the other hand, Alveolata, Chloroplastida, Discosea, Holozoa, Rhizaria, Stramenopiles, and Tublinea were the dominant non-fungal eukaryotic classes. The highest abundance and major contribution to interactions by Sordariomycetes and Holozoa in the fungal and non-fungal eukaryotic communities respectively assert their contribution in the respective communities in the compost environment. The findings of this study extend the understanding of the evolution of fungal and non-fungal eukaryotes as well as key players in communal interactions during composting.

The authors wish to thank the Pan African Network for Bioinformatics Training (H3ABioNet) through Caleb Kibet and Andrew Espira of the International Centre of Insect Physiology and Ecology (ICIPE) node for providing necessary computational infrastructure and invaluable support to bring this project to a good end. The authors acknowledge the support received from SysCom Kenya research assistant James Karanja during fieldwork.

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16 Jan 2023

PONE-D-22-30019Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks.PLOS ONE

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Reviewer #1: Page 16 line 190, 191 and 290: during composting, we know that the carbon content is decreasing throughout the composting, while nitrogen and phosphorus are increasing, while the temperature is increasing from the beginning until the maturation phase, I think it is contradictory to say that there is a positive correlation between temperature and carbon. so how could the authors explain the positive correlation between temperature and carbon?

Page 16 line 202, 204 and 206 : what do the authors mean by and 84 days of composting ??? it doesn't make sense ? the authors should change these sentences and review the whole document ?

Page 18 line 243 and 253; are these a title or what?

Conclusion: authors should improve the conclusion to be more attractive.

English should be improved.

I think this article is lacking in novelty.

The quality of all figures is poor and unreadable, the authors should improve the quality of all figures.

Reviewer #2: The present manuscript investigates the structure and contribution of fungal and non-fungal community during thermophilic composting, through the implementation of four experimental schemes (Lantana, Tithonia, Grass, mixture). The research is in general interesting. The abstract section contains information which belong to the materials methods. Lines 30-35 should not include methodology but the actual findings of the research. Authors state that findings of this study unravel influence composting materials on the eukaryotic community structure but this is not supported in the previous lines (1-41). The introduction section is very general; it includes many references but very little information on the actual subject. In lines 70-72 is reported that Lantana camara and Tithonia diversifolia are phytochemicaly complex and the Lantana contain complex polymers without further details. Authors should consider the structure and the content of the introduction, which should increase and contain useful and specific scientific information. Please clearly report (using a plain table) the physicochemical characteristics of the compost schemes. All figures have poor resolution and should be resized. Lines 188-192 are more like a general statement and not a detailed presentation of the results, where no correlation is reported and the same apply for all the section of the results. Please include the relative abundance of the main fungal classes and please do the same for the non-fungal communities. Lines 245-249, Lines 256-258 are not presenting any results. Lines 271-271 are confusing. What the authors mean by ‘’preferred less ammonia and responded positively to decrease in other....states of the compost’’? There is no discussion on PCA and on the statistical analysis. The conclusion section is very general.

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 10.1371/journal.pone.0286320.r002 Author response to Decision Letter 0 Submission Version 1

27 Feb 2023

I would like to appreciate the opportunity to submit a revised version of the manuscript entitled “Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks” to be considered for publication in your Journal as an original article. Kindly also note that the referred line numbers are on the “Revised Manuscript with Track Changes” files.

We highly appreciate and welcome your comments and those of the reviewers and have addressed the points raised during the review process as follows;

Reviewer Comments:

Reviewer #1:

Reviewer comment No. 1: Page 16 line 190, 191 and 290: during composting, we know that the carbon content is decreasing throughout the composting, while nitrogen and phosphorus are increasing, while the temperature is increasing from the beginning until the maturation phase, I think it is contradictory to say that there is a positive correlation between temperature and carbon. so how could the authors explain the positive correlation between temperature and carbon?

Authors’ response:

The two variables tended to increase and decrease together, thus making the correlation value positive. It is true that during composting, carbon decreases. However, temperature also decreases during the composting period. Of all the sampling days, the highest temperature and carbon were recorded at 21 days of composting, with both variables decreasing along the composting periods. Plausible reasons for the positive correlation of the two variables are in lines 368 to 372. The authors have enriched this manuscript by adding table 2 and lines 218-221 containing data on the trends of physical-chemical variables along the composting process to provide more information.

Reviewer comment No. 2: Page 16 line 202, 204 and 206: what do the authors mean by and 84 days of composting ??? it doesn't make sense ? the authors should change these sentences and review the whole document ?

Authors’ response:

This statement was used to indicate the number of days (sampling times/points) from the time of heaping compost to maturation within 84 days. Nonetheless, the word has been rephrased to read as “day 84 of composting” to qualify the statements throughout the manuscript.

Reviewer comment No. 3: Page 18 line 243 and 253; are these a title or what?

Authors’ response:

The titles have been revised on page 16 lines 306-308 and 320-322 respectively to read as “Unique fungal and non-fungal eukaryotic taxa within compost environment” and “Fungal and non-fungal community interactions within compost environment.”

Reviewer comment No. 4: Conclusion: authors should improve the conclusion to be more attractive.

Authors’ response:

The conclusion has been rewritten as advised.

Reviewer comment No. 5: English should be improved

Authors’ response:

The authors have read through the manuscript and where applicable, the most appropriate adjectives have been adopted to improve the manuscript as recommended.

Reviewer comment No. 6: I think this article is lacking in novelty

Authors’ response:

The authors believe that the novelty of this article lies in lines 56-59 , 71-76 and 85-88. Lines 64-70, and 90-96 have been added to provide more information on the novelty of the study.

Reviewer comment No. 7: The quality of all figures is poor and unreadable, the authors should improve the quality of all figures

Authors’ response:

The figures have been improved for better resolution.

Reviewer 2

Reviewer comment No. 1: The present manuscript investigates the structure and contribution of fungal and non-fungal community during thermophilic composting, through the implementation of four experimental schemes (Lantana, Tithonia, Grass, mixture). The research is in general interesting. The abstract section contains information which belong to the materials methods. Lines 30-35 should not include methodology but the actual findings of the research.

Authors’ response:

The abstract has been revised in lines 31-39 to describe the main objective(s) of the study, explain how the study was done, and a summary of the most important results and their significance. Methodological details have been removed as advised.

Reviewer comment No. 2: Authors state that findings of this study unravel influence composting materials on the eukaryotic community structure but this is not supported in the previous lines (1-41).

Authors’ response:

The influence of composting material on eukaryotic community structure is shown in lines 29-31 and results in lines 36-44.

Reviewer comment No. 3: The introduction section is very general; it includes many references but very little information on the actual subject.

Authors’ response:

More specific information on the influence of various feedstocks on eukaryotic communities has been added and appropriately cited in lines 64-96 within the manuscript.

Reviewer comment No. 4: In lines, 70-72 is reported that Lantana camara and Tithonia diversifolia are phytochemicaly complex and the Lantana contain complex polymers without further details.

Authors’ response:

Further details regarding the phytochemical complexities of Lantana and Tithonia have been added in lines 82 to 84 within the manuscript.

Reviewer comment No. 5: Authors should consider the structure and the content of the introduction, which should increase and contain useful and specific scientific information.

Authors’ response:

More specific information on the influence of various feedstocks on eukaryotic communities has been added and appropriately cited in lines 64-96 within the manuscript.

Reviewer comment No. 6: Please clearly report (using a plain table) the physicochemical characteristics of the compost schemes.

Authors’ response:

Table 2 and lines 218-221 have been added to the results section for clarity.

Reviewer comment No. 7: All figures have poor resolution and should be resized.

Authors’ response:

The figures have been improved for better resolution.

Reviewer comment No. 8: Lines 188-192 are more like a general statement and not a detailed presentation of the results, where no correlation is reported and the same apply for all the section of the results.

Authors’ response:

Detailed results (tables 2) and explanations have been added in lines 218-234 to enhance the manuscript

Reviewer comment No. 9: Please include the relative abundance of the main fungal classes and please do the same for the non-fungal communities.

Authors’ response:

This has been added in text lines 271-279 and tables 3A and 3B to provide the computed relative abundances to the manuscript.

Reviewer comment No. 10: Lines 245-249, Lines 256-258 are not presenting any results.

Authors’ response:

Lines 308-315 and 322-332 have been added to improve the result presented.

Reviewer comment No. 11: Lines 271-271 are confusing.

Authors’ response:

The lines (345-346) have been rephrased for clarity.

Reviewer comment No. 12: What the authors mean by ‘’preferred less ammonia and responded positively to decrease in other....states of the compost’’?.

Authors’ response:

This implies that they thrive under lower levels of ammonia. Nonetheless, the line/s (345-346) has been rephrased for clarity.

Reviewer comment No. 13: there is no discussion on PCA and on the statistical analysis

Authors’ response:

Discussion on PCA has been added to lines 366-368.

Reviewer comment No. 14: The conclusion section is very general

Authors’ response:

The conclusion has been rewritten as advised.

We hope that we have adequately responded to all the comments raised regarding this manuscript in readiness for its publication in your journal.

 10.1371/journal.pone.0286320.r003 Decision Letter 1 Bhattacharjya Sudeshna Academic Editor 2023 Sudeshna Bhattacharjya This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

5 Apr 2023

PONE-D-22-30019R1Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocksPLOS ONE

Dear Dr. Felix Matheri,

Thank you for submitting your manuscript to PLOS ONE. Reviewers have submitted their comments and we are positive about the manuscript. However, the manuscript still needs some minor revisions before it can be processed further. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Sudeshna Bhattacharjya, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you very much for the authors efforts to respond to my comments, but in the still you should improve your manuscript, there are some further comments.

line 75: Please correct "in invitro" by dropping "in".

Line 113,145: write "icipe" in capital letters.

Table 2: Authors should indicate the meaning of each symbol and the unit: for example, Carbon (C), ditto for all others (N, P .....).

line 261: regarding the fungus class nomenclature, the authors should remove the italic character style. please review the overall manuscript

You still need to demonstrate the novelty of your manuscript, it is not clear.

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: Saloua Biyada

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

 10.1371/journal.pone.0286320.r004 Author response to Decision Letter 1 Submission Version 2

26 Apr 2023

I would like to appreciate the opportunity to submit a revised version of the manuscript entitled “Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks” to be considered for publication in your Journal as an original article. Kindly also note that the referred line numbers are on the “Revised Manuscript with Track Changes” file.

We highly appreciate and welcome your comments and those of the reviewers and have addressed the points raised during the review process as follows;

Reviewer Comments:

Reviewer #1:

Thank you very much for the authors efforts to respond to my comments, but in the still you should improve your manuscript, there are some further comments.

Reviewer comment No. 1: line 75: Please correct "in invitro" by dropping "in".

Authors’ response:

The word ‘in’ has been dropped and replaced with ‘under’.

Reviewer comment No. 2: Line 113,145: write "icipe" in capital letters.

Authors’ response:

This is acknowledged and changed throughout the manuscript.

Reviewer comment No. 3: Table 2: Authors should indicate the meaning of each symbol and the unit: for example, Carbon (C), ditto for all others (N, P .....).

Authors’ response:

This has been corrected as advised.

Reviewer comment No. 4: line 261: regarding the fungus class nomenclature, the authors should remove the italic character style. please review the overall manuscript.

Authors’ response:

This has been changed throughout the text as advised.

Reviewer comment No. 5: You still need to demonstrate the novelty of your manuscript, it is not clear.

Authors’ response:

The authors aver that the novelty of the study is on lines 25-29, 72-80. The rationale for this study is articulated within the manuscript in lines 93-101, where the justification for studying the diverse green materials has been presented “These materials have, however, been reported to have antimicrobial activity in in-vitro experiments. Furthermore, the phytochemical analysis has shown differences in their complexities, thus possibly requiring various periods to break down. Therefore, it is necessary to understand these materials' influence on the biological and physical-chemical stability of compost” and lines 65-75 “Some of these materials have been reported under invitro studies as having inhibitory properties on fungal and non-fungal organisms [13, 14]. The phytochemical complexities of Lantana camara and Tithonia diversifolia have been previously studied. Lantana camara has been reported to contain more complex polymers and thus possibly requires more microbial categories to break down into agriculturally useful material. For example, the phytochemical composition analysis of L. camara showed 23.3% crude fiber, 26% cellulose, 16.2% lignin, and 21% hemicellulose, while T. diversifolia contains 11.2%, 17%, 7%, and 16% of these elements respectively [15, 16, 17]. Furthermore, the different complexities of the composting material could require assorted composting times to produce a biogeochemically stable product. It is, therefore, necessary to evaluate the influence of composting time on the biological and physical-chemical quality of compost”

The importance of Eukaryotes in the ecosystems and their limited study is enumerated in lines 51-56 “Though less studied, eukaryotes are an important microbial category in the ecosystem with diverse feeding and genetic guilds [6, 7]. Eukaryotes degrade recalcitrant material such as Carbon-rich polymers, thus contributing to nutrient cycling in the compost environment [8]. The complex structure and function of a large number of compost microorganisms are directly affected by various environmental factors such as temperature, moisture, Carbon/Nitrogen ratio, and pH, among others”.

The use of next-gen sequencing to study eukaryotes is justified in lines 124-130 “Numerous studies have assessed the microbial prokaryotic community in the composting process using culture-dependent and culture-independent methods [2, 4, 6, 10]. However, there is still a limited understanding of eukaryotic communities’ structure in the composting process, concerning the composition and complexity of the composting materials especially utilizing high-throughput sequencing technology. The effect of the Lantana camara and Tithonia diversifolia on microbial community structure in complex ecosystems such as the compost environment has not also been done, despite their widespread adoption by farmers as soil nutrient amendment materials”.

Moreover, lines 29-30, and 57-59 have been added to increase clarity.

Reviewer 2

Reviewer #2: (No Response)

Authors’ response:

The authors appreciate the great input from the reviewer.

We reiterate our appreciation to the reviewers for their kind comments which have helped improve the manuscript and hope that we have adequately responded to all the comments raised regarding this manuscript in readiness for its publication in your journal.

Thank you for your consideration.

Yours Sincerely

Felix Matheri

Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University,

P.O Box 30772-00100 Nairobi.

Submitted filename: Response_to_Reviewers.docx

 10.1371/journal.pone.0286320.r005 Decision Letter 2 Bhattacharjya Sudeshna Academic Editor 2023 Sudeshna Bhattacharjya This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

15 May 2023

Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks

PONE-D-22-30019R2

Dear Dr. Matheri,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sudeshna Bhattacharjya, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Saloua Biyada

**********

 10.1371/journal.pone.0286320.r006 Acceptance letter Bhattacharjya Sudeshna Academic Editor 2023 Sudeshna Bhattacharjya This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

22 May 2023

PONE-D-22-30019R2

Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks

Dear Dr. Matheri:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Sudeshna Bhattacharjya

Academic Editor

PLOS ONE