Succession and Catabolic Properties of Fungal Community During Composting of Fruit Waste at Sub-Tropical Environment

A comprehensive profile of structural and functional dynamics of the fungal community during fruit waste composting was investigated. For this purpose, fruit waste was composted in a windrow setup in a sub-tropical environment for 146 days. Representative samples were collected at varied range of temperatures during composting and employed to physicochemical and microbiological analysis. Time-series data revealed that variation in fungal load is temperature dependent that influenced morphotypes’ shifts during different stages of composting. Shifts in abiotic factors, availability of accessible nutrients, water loss, pH and electrical conductivity (EC) participated in the transition of community and compost maturity. Fungal community shifts were studied using a culture-based method, denaturing gradient gel electrophoresis (DGGE) and Illumina sequencing. The Ascomycota were the most common phylum recovered using culture-based and DGGE methods. However, Illumina sequencing revealed a predominance of phylum Zygomycota (Mortierella sp, Rhizomucor sp), which had a low representation in the culture-based study, and phylum Basidomycota (Coprinopsis sp, Coprinellus canistri & Coprinus cordisporus), which was absent in the culture-based study. In contrast, Aspergillus fumigatus (ADIF1), which was found to be the most promising cellulase and pectinase producer at higher temperatures in culture-based studies, had a very low representation in molecular studies. The current findings imply that compost is a habitat for a diverse fungal population, it should be investigated using molecular approaches to study in-depth fungal ecology. Furthermore, the transformation of fruit waste into seed germination-friendly compost via a diverse fungal community yields an efficient organic fertilizer.


Introduction
Management of biowaste is a huge problem in low-and middle-income countries. These countries generate around 54% of biowaste of their total waste [1]. Open dumping of these wastes in landfill sites results in air and ground water pollution through leaching which poses a high risk to human health. Composting is a cost-effective and easy method to manage waste in developing countries. It not only reduces the waste volume but also provides organic fertilizer that could be used to enhance soil fertility [2]. The composting process is a combined activity of bacteria, fungi and actinomycetes. Usually, bacterial community outnumbers the fungal community during the mesophilic and thermophilic phases of composting [3][4][5], owing to their quick generation time. The other reason that favors bacterial growth is the physicochemical properties of feedstock. For example, the presence of Lactobacillus in the initial stage indicates influence of low pH on their growth [6]. Similarly, low pH also favors the growth of yeasts in early phase of composting [7]. But strikingly, fungi being the efficient degraders of recalcitrant substances in the later stages of composting have not been thoroughly studied earlier and its importance has remained neglected. Recently, several studies attempted to study fungal succession by incorporating molecular methods like denaturing gradient gel electrophoresis (DGGE) and pyrosequencing [7,8]. These studies revealed that the existence of standard microbiota during composting is not possible since it's a diverse process that is highly dependent upon feedstock used and the technique of composting applied. It does not operate in standard environmental conditions but the variation in physical and biological factors fairly follows the same trend [9]. It has been stated that different composting methods, waste sources, and environmental parameters result in different microbial communities, which is why it is critical to isolate and study their participation to generate high-quality compost [7,[10][11][12][13]. Considering these facts, in this study we planned to use fruit waste as the main feedstock for composting. Fruit waste contains high content of nitrogen (N) and other macronutrients like phosphorous (P), potassium (K), calcium (Ca) and magnesium (Mg) which upon degradation could play role in enrichment of nutrient deficit soils. These macronutrients play a major role in plant growth and addition of macronutrients in to soil increases the abundance of saprophytic fungi [10].
The enrichment of the soil can be done through composting of these organic waste such as fruit peels, soybean residues and food waste since they are easily available and contain high nutrients (N,P,K). But most of the time these wastes are not utilized effectively and are disposed of through burning. To the best of our knowledge, no study in Southern Pakistan was conducted to provide a comprehensive idea of fruit waste composting under sub-tropical conditions and the microorganisms involve during the process of degradation. Karachi's climate is classified as 'BWh' (hot arid climate) by the World Koppen-Geiger climate classification, having hot summers and warm winters and low yearly precipitation. However, as a coastal city, it has a high level of humidity i.e. 59.7%, with an average outdoor temperature of 32 °C in the summer [11]. Since local environmental conditions play a very important role with the feedstock and physical parameters used, we were aiming to observe the variation in fungal community during composting under such conditions. As many developing countries are in the subtropical zone, with hot and humid summers and moderate winters [12], this research was carried out in Karachi, Pakistan, which has similar climatic conditions. In this way, it can serve as a framework for other countries to follow in order to convert waste into usable form.
In this study, microbiological and analytical methods were used to access the biodiversity of fungal communities. To achieve this aim, following objectives were targeted, (a) to assess the rate of biodegradation and time required to compost maturation; (b) to monitor environmental parameters and study its transitional effect on fungal diversity; (c) to study structural and functional composition of resident fungal communities and their dominancy over various phases of composting and (d) to assess phytotoxicity of mature compost.

Composting Setup and Physical Parameters
The study followed a windrow setup for the degradation of fruit waste through composting. Fruit waste was used as a key substrate along-with green leaves, fresh grass clippings and untreated sawdust to achieve a carbon to nitrogen (C/N) ratio of 31:1. It was achieved through a volume-based ratio calculation of each substrate. These substrates were also amended with garden soil that acts as starter culture in composting. Prior to composting, each layer of substrate was moistened enough to adjust the moisture content to 60-65%. This setup was done on an unpaved ground in green house conditions at the backyard of the department of Microbiology, University of Karachi (24°56′ 24′ N 67°7′ 6′ E) for a period of five months, i.e., March to August 2017 and exposed to environmental temperature ranging from 29-36 ºC. This is a subtropical climate with high temperatures averaging around 32 °C during the summer. The pyramid shape windrow pile (105 cm long × 85 cm wide × 110 cm tall) was aerated and moistened periodically based on its temperature and moisture profile which was monitored through mercury thermometer and oven drying method, respectively [7]. Temperature and moisture content was monitored from different sections within a pile; surface (10 cm from top) and center (55 cm from top) of pile.

Sampling Strategy and Chemical Analysis:
Efficient compost has certain established physicochemical conditions. Samples from different positions of compost pile were collected to make a composite representative sample of each sampling day and then it was subjected to physicochemical and fungal community analysis. All the analyses were performed in triplicates. Total carbon (TC) was determined gravimetrically after ignition in oven at 750 °C for 2 h and Total nitrogen (TN) was estimated using Kjeldahl method [13]. C/N ratio was calculated as the quotient obtained after dividing carbon content to nitrogen content [14]. The pH and EC were measured using pH and conductivity meter (Hach, USA). Macronutrients (P,Ca,Mg) were quantified through atomic absorption spectrophotometer using an acid-treated compost samples [15].

Culture-dependent Fungal Community Analysis
A suspension of compost using 10 g of sample and 100 ml of phosphate buffer saline (PBS) was prepared and shaken for 30 min to homogenize the sample. The viable fungal fraction of each sample was enumerated using ten-fold serial dilution and 100 µl of serial dilutions (10 -1 to 10 -4 ) were inoculated on sabourauds dextrose agar (SDA, sigma UK) medium. Inoculated plates were incubated in triplicates at five different temperatures (25 °C, 37 °C, 45 °C, 50 °C and 55 °C) to have complete mesophilic and thermophilic profile of fungal community. In case of mesophilic fungal cultivation (25 °C, 37 °C), the incubation time was 5-7 days till no increase in number of fungal cultures was observed. For thermophilic fungi (45 °C, 50 °C and 55 °C) the incubation time was 3-5 days. This difference of incubation time between mesophilic and thermophilic fungal isolation was due to exposure of growth media to high temperature causing its dehydration. The viable fungal population was expressed in CFUg −1 of compost sample. The fungal colonies that differ in macroscopic characteristics were sub-cultured on SDA and were used for morphological grouping [16] to estimate their diversity and then followed with microscopic observations to confirm the isolates. The fungal isolates belong to different genera were again sub-cultured and purified for molecular identification based on its sequencing. Prior to DNA extraction, these distinct fungal colonies were treated with liquid nitrogen and then genomic DNA extraction was carried out as described elsewhere [17]. Amplification of ITS1-5.8S-ITS2 region of fungal rRNA gene was performed using primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') [18]. The reaction proceeded for 35 cycles with denaturation at 95 °C for 30 s, annealing at 55 °C for 60 s and elongation at 72 °C for 120 s. Amplified PCR products were purified using QIAquick PCR Purification Kit following manufacturer's instructions (Qiagen, UK). Sanger dideoxy sequencing of purified amplicons were carried out via Macrogen. Accession numbers were obtained from National Center for Biotechnology Information (NCBI) database.
Amplicons from three replicates were pooled and purified using a Qiagen purification kit according to the manufacturer's instructions. DGGE was performed according to the protocol described elsewhere [20]. The position and intensity of the bands was quantified using Phoretix ID Advanced V5.00 software (Nonlinear dynamics, UK). In order to standardize and allow comparisons to be made between different gels, pooled PCR products amplified from the DNA of Penicillium chrysogenum, Rhizopus oryzae, Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus were included as a species ladder on every gel.

Culture-independent Fungal Community Analysis via Illumina Miseq
Microbial community DNA was extracted from 1 g mature compost (week 20) using DNeasy Powersoil kit (QAIGEN) according to manufacturer's instructions with modifications. Vortexing time was increased for proper lysis step i.e., 20 min and centrifugation was done at 12,000 rpm for 2 min Eluted DNA was visualized using UV transilluminator (UVP's patented Benchtop Transilluminator). Gel Electrophoresis was performed in 1% agarose gel stained with 1.5 μl ethidium bromide. Wells were loaded with 3 μl of DNA sample. Gel was run for about 15 min along with DNA ladder at 100 V. Samples were stored at − 20 °C till required. Extracted DNA samples were sent to BGI, Hong Kong for library construction, Internal Transcribed Spacer (ITS) amplicon sequencing and bioinformatics analysis. Fungal ITS 1 region sequencing was done by using illumina MiSeq system that generates 300 paired ends (50,000 TAGS).

Catabolic Profiling
Selected fungal isolates were also screened for their cellulase and pectinase activity at mesophilic (30 °C) and thermophilic (50 °C) temperatures. Well diffusion method and agar plate method were employed to determine cellulolytic and pectinolytic activities, respectively. In well diffusion method, fungal cultures were grown on carboxymethyl cellulose (CMC) broth [21] at 150 rpm in a shaking incubator for 72 h and harvested by filtration through Whatman no. 1 filter paper that resulted in cell free culture supernatant. Test media (cellulose 1%, Congo red 0.5%, agar 1%) was bored and 40 µl of cell free culture supernatant was added and incubated for 24 h. Plates were stained with Congo red dye (1%) for 15 min followed by de-staining with 2X NaCl for 15 min [22]. The halo around well is indicative of cellulolytic activity. For quantitative analysis of cellulase, cell free culture supernatant was used as a crude enzyme to perform endoglucanase assay [21]. Crude enzyme (50 µl) was added to equal volume of 1% (w/v) CMC in 50 mM sodium citrate buffer, pH 4.8. The tubes were incubated for 30 min at respective temperatures (30 °C and 50 ºC) followed by the determination of reducing sugar. One unit of endoglucanase activity was defined as the amount of enzyme that can hydrolyze CMC and release one micromole of glucose under standard assay conditions [21]. In agar plate assay, isolated fungal colony was spotted through needle on media plate containing minimal salt medium with 1% pectin [23] and left for overnight incubation. Pectinolytic fungi were identified through flooding the incubated plates with iodine crystals resulting in the production of clear halo around colonies.

Phytotoxicity Assessment
The maturity of compost was assessed by carrying out phytotoxicity assay as described by Haq et al. [15] and Ansari et al. [24] using green mung bean (Vigna radiata) as test seeds. For this purpose, seed germination was performed in a petri dish lined with sterilized cotton and filled with aqueous extract of compost (5 ml). This compost extract was obtained by mixing 10 g of compost sample with 100 ml of distilled water, shaking it for 30 min at room temperature for homogenization and then filtering it through Whatman no. 1 filter paper. In control plates, autoclaved distilled water (5 ml) was used. Then six mung beans were placed in each plate and incubated in dark for five days. After incubation time, germination index (GI) was calculated by the root length and the percentage of germination of selected test plant seeds compared to a control using following formulae.

Statistical Analysis
Descriptive statistics (Mean and standard deviation) were calculated for triplicates of each sample by using SPSS software (IBM SPSS STATISTICS 20). The observed data were also subjected to one-way analysis of variance (ANOVA) to determine the differences of physicochemical and microbiological analysis in each sample during composting. Significant acceptance was considered at P-value < 0.05. Principal Component Analysis (PCA) of bands on DGGE gel was performed using Multi-variate Statistical Package (MVSP) version 3.22 (copyright © 1985-2003 Kovach computing services).

Relative seed germination (%) =
Number of seeds germinated in extract Number of seeds germinated in control × 100 Relative root elongation (%) = Mean root length in extract Mean root length in control × 100 Germination Index (%) = Relative seed germination × Relative root elongation 100

Changes in Physicochemical Parameters
Temperature of compost pile (surface and center) and environment was monitored throughout the composting process that started on zero day and ended on 146th day. Changes in temperature profile were depicted in Fig. 1. It shows that as compared to the ambient temperature i.e. 28 °C, the temperatures of center and surface of pile was started to increase overnight and peaked to 42 °C (surface) and 49 °C (center) on 7th day indicating thermophilic phase. This rise in temperature was due to quick decomposition of easilybiodegradable organic compounds [3]. With progressive degradation, temperature of the compost pile gradually decreased and thereafter remained consistent with time to ambient temperature. As shown in Fig. 2, the process was started from 67% of water content and ended at significant loss to 22.4% (surface) and 31.5% (center) by the end of composting. The difference in moisture loss between surface and center of pile was due to more exposure of surface heap to sun that led to evaporation [25], while water in the center remain trapped. The variation in pH and EC throughout the composting process is shown in Fig. 3. In the case of pH, the feedstock was slightly acidic initially, i.e., pH 6.0 due to acidic nature of citrus fruits waste. As acidic feedstock underwent degradation, rise in pH was observed. This significant rise (P < 0.05) to alkaline pH 8.1 was observed on 7th day, possibly due to protein hydrolysis or ammonia formation. From 7th day onwards, pH declined to neutral (7.0) and then stabilized at pH 6.0 till curing. In the case of EC, at the time of piling, the compost conductivity was 0.16 mScm −1 and with the progressive degradation it ended at 0.26 mScm −1 with a significant increase (P < 0.05).
The changes in C/N ratio, and macronutrients are tabulated in Table 1. Composting was started with a C/N ratio of 31:1 which is an optimum range for composting. Over the composting period, C/N ratio declined consistently and significantly (P < 0.05) and ended at 19:1. Variations in macronutrients content was evident. As in the case of Ca and Mg a significant increase (P < 0.05) was observed in samples collected from initial and final stages of composting. Whereas P content was found to be consistent throughout the process of composting.
The efficiency of cured compost can be assured by the physicochemical parameters. The overall results suggest that thermophilic range was obtained just in the first week of degradation, the same time pH rose as well. The first seven days also showed significant rise in N content along with other macronutrients. The same period showed a drop in C content, C/N ratio and organic matter. Decrease in organic matter suggests the degradation of waste material in compost that led to the release of nutrients. The heat generation during active phase of composting and presence of optimum concentration of nutrients in matured compost indicated the efficiency of compost.

Changes in Fungal Communities During Composting via Culture-Dependent Technique
Fruit waste was primarily degraded by filamentous fungi, which could be grouped based on colonial characteristics. Therefore, relative progression of each morphotype was calculated by counting the distinctive colony in each sample at various time intervals and at various incubation temperatures. The dynamics of fungal communities at all incubation temperatures are demonstrated in Fig. 5  The results proposed that variation in colonial characteristics of fungal isolates were dependent on the temperature and the local abiotic conditions of the compost.
These morphotypes were then analyzed under a microscope and classified into genera. With reference to Zafar et al. [26], this grouping and probable identification of isolates was done based on culture and microscopic morphology. Among those genera, samples have been dominated by Aspergillus spp. for the entire 146-day composting period (Fig. 6). This demonstrated the fungal community's active participation in fruit waste degradation through composting.

Changes in Fungal Community During Composting via Culture-Independent Technique
DGGE revealed that in the compost, the fungal population diversity was moderate initially when the pile was first formed. After week 4 and week 8, the community profile was very low that could be due to higher temperature, where bacteria play a major role in the degradation of organic waste (Fig. 7). As the temperature went down and compost got mature, the diversity of fungal community has increased. PCA analysis of Boolean data (presence/absence of bands on DGGE, 0 or 1), confirms the succession of fungal community dynamics from week 0 to week 20. The PCA analysis clearly demarcates the three samples into thermophilic, mesophilic, and curing phases of compost ( Fig. 8 and 9). Three bands were recovered from first two stages (week 4 and week 8) at different positions and thirty-three bands were recovered in the last sample suggesting the richness of community.  to Penicillium chrysogenum only appeared in samples at mesophilic temperature range i.e., week 0 and week 20. Band 3 was constantly present throughout the composting process with thickness variability, which could be attributed to its abundance. Interestingly, the fungal genus corresponding to this band was either not or hardly recovered through culture-based technique implied. Band 4 corresponds to Rhizopus oryzae which was frequently recovered in culture-based technique but appeared in low abundance through DGGE (Fig. 7). Another interesting observation revealed through DGGE analysis is that the species recovered regularly through culture-based technique were not prominent in the molecular analysis. This could be because of their high germination rate exploited during culturing or biasness of ITS primers during community PCR. DGGE also revealed that there are many other species present that were not recovered through conventional culturing techiques.

Biodiversity of Fungal Communities Associated with Fruit Waste Composting by Illumina Miseq
A total of 131,853 sequences were obtained after removing all poor quality and short reads data and were clustered into 47 different OTUs at 97% sequence similarity. In order to understand the complexity of sample, alpha diversity was applied through indices such as observed species ( Figure  S2 supplementary file) Chao1, Shannon-index and Simpson (Table S2 supplementary file). Complexity of sample is proportional to all indices except that of Simpson value. Species richness of community is usually reflected by observed species and Chao1 value. Shannon index together with Simpson value provide an idea about species diversity in community that ultimately depends on species richness and evenness. The rarefaction curve indicated the produced data was enough to cover all 47 species in fungal community. The straightening of curve suggested, data produced was enough to study diversity of sample.
Species annotation showed that sequences were associated with three phyla including Zygomycota, Basidiomycota and Ascomycota and among phyla, Zygomycota was found as the most dominant one with relative abundance of 52.16% (Fig. 10). On species level, Mortierella sp. and Rhizomucor sp. were identified. Basidiomycota revealed as the second dominant phyla with relative abundance of 46.04%, identified species were Coprinopsis sp., Coprinellus canistri and Coprinus cordisporus. Results indicated less abundance (0.69%) of Ascomycota detected with Scutellinia sp., Schizothecium carpinicola, Penicillium sp., Preussiaterricola, Arthrobotrys thaumasia, Zopfiella sp., Montagnulaceae sp., Trichoderma longibrachiatum, Aspergillus sp., Dactylella sp., Monascus purpureus, Myrmecridium schulzeri, Emericella sp. and Fusarium sp. Phylogenetic tree at genus level from different phyla is shown in Fig. 11. The identified illumina sequences were submitted to NCBI GenBank for accession numbers as mentioned in (Table S3 Supplementary file).

Catabolic Profiling of Fungi
Since, the compost pile has been made from fruit waste, leaves, and saw dust, it became a good source of pectin and CMC was used as carbon source for screening of cellulase producers. Cellulolytic activity of all the fungal isolates was determined by its ability to degrade β-D-glucan in medium. At mesophilic temperature, fungal isolates that possessed cellulolytic activity were Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium sp. and Cladosporium sp. Whereas, only Aspergillus fumigatus (ADIF1, ADIB8 and ADIK2) exhibited cellulolytic activity at thermophilic temperature. The highest cellulolytic activity was shown by ADIF1 (A. fumigatus) at 50 °C, i.e., 4.40 IUml −1 , via Endoglucanase assay. The cellulolytic activity of fungal strains was much higher at thermophilic temperature compared to mesophilic temperature. To our understanding, high temperature was optimum for the growth and secretion of cellulases by fungal strains.
Pectinase production was observed as halo around fungal colony due to depolymerization of pectin in growth media by exposing with iodine crystals. In this study, Aspergillus flavus and Penicillium sp. were observed as pectinase producers at 30 °C and Aspergillus fumigatus exhibited pectinolytic activity at 50 °C.
The results presented in Table 2 indicated the importance of genus Aspergillus for their hydrolytic activity and their detailed study could result in a development of better strains that can effectively degrade complex substances and has diverse industrial applications.
All the representative fungal isolates that were selected and subjected to enzymatic analysis were preferred for identification through amplification of ITS gene (Table supplementary file). DNA of Cladosporium sp. could not be recovered that is why its molecular identification was not performed. In general, the results suggested that temperature variation in the incubation and composting itself favors the growth of certain species at specific time intervals of composting process. Investigation at five different temperatures, helped the comprehensive exploration of microbiological niche found in compost.

Phytotoxicity of Compost
GI was calculated by combining the measure of relative seed germination and relative root elongation. In this study, maturity of fruit waste compost was compared with the cattle manure and chemical fertilizer based on GI of Vigna radiata. The results obtained were 83.3%, 70.4% and 27.3% respectively. GI of fruit waste compost was higher than chemical fertilizer (P > 0.01) and animal manure, hence indicating its maturity and absence of phytotoxicity (Table 3).

Discussion
Microbial communities are major contributors of biodegradation of waste. The microbial dynamics depends a lot on the management, the environmental conditions in which the composting process takes place and the organic substrate under study. In this study, fungal community and their enzymatic capability and the effect of abiotic parameters on the community during fruit waste composting were investigated using culture-dependent and culture independent microbiological techniques.
Starting with physicochemical parameters, temperature being a critical parameter during composting, affects the waste degradation and its biological activity [26]. All phases of windrow composting in relation to temperature were observed. The high temperature lasted a few days before gradually declining to a mesophilic stage and maturation stage, where the pile temperature stayed constant over time as it approached ambient temperature. These temperature changes were caused by microbial metabolism, which supports a shift in the microbial community and was in accord with the previous studies [15,26,27]. Moisture content was gradually decreased over time despite repetitive water addition. Since water is required for microbes as a medium of nutrient transportation, therefore, water loss indicates intense microbial activity. This results in heat generation that led to a rise in temperature which eventually facilitates the decline in moisture content [2,28]. Several researchers [29][30][31] have also reported the same trend and claimed that a gradual decrease in moisture content is indicative of stable and mature compost. The results showed that mature compost was stabilized at pH In accordance with another study [28], volatilization of ammoniacal nitrogen and release of H + ions during microbial nitrification process contribute to low pH. It has also been reported that pH is not an absolute indicator of compost maturity; it primarily depends upon the type of feedstock and method used for composting [32]. EC has a great impact on compost application to agricultural land; it could act as a limiting factor during germination of seed and growth of plant [33]. The data in our study expressed several gradual fluctuations over time. However, the observed values were never below the initial reading of conductivity. It has been reported that an upward trend of conductivity during composting process was observed due to release of ions like Ca 2+ , Mg 2+ , Na + , K + and PO 4 −2 [31,34]. Likewise, the sudden decline in EC corroborated with another study where it was suggested that leaching of mineralized ions after water addition or formation of soluble salt complexes due to humification results in less number of free ions thereby a decline in EC is observed [27]. This idea has also been confirmed in a study [35] where compost leachate has high conductivity. In present study, the finished compost has EC value of < 3 mScm −1 indicating safe application of compost to soil [15]. C/N ratio not only constitutes a significant parameter that indicates the extent of complete biotransformation but also the availability of nitrogen for plants [36]. Over the composting period, C/N ratio declined consistently and significantly (P < 0.05) and ended at 19:1 The drop in C/N ratio is relevant with the increase in N, which is because of mineralization of nitrogenous compounds, and decrease in C, due to production of CO 2 during organic matter decomposition [33]. These findings are in consent with the findings of Carmo et al. [37] where a correlation in the increase in inorganic matter loss with the exhaustion in C/N ratio was observed. A possible explanation is the accelerated activity of microbial decomposition. In the present investigation, the C/N ratio of mature compost was found to be in the optimum range i.e. < 20:1 that can enable the availability and accessibility of nutrients for plants upon application [4].
The nutrient concentration of the final compost varies with the feedstock and method applied for decomposition [36]. In this study, major macronutrients; P, Ca and Mg were estimated. Individually, Ca and Mg showed escalation, as the process progressed till the end and these observations are also in agreement with the reported results [15,38]. On the contrary, P content escalated (P < 0.05) on the 7th day, remained static (P > 0.05) till day 76th and then took a significant decline (P < 0.05) on the 146th day. This decrease of nutrients during 76th day to 146th day of composting could be a result of leaching from the degraded waste [39]. The other possible explanation behind this sudden increase and decrease can be related to pH. As reported previously [33], the P content is very sensitive to pH; a slight deviation from its optimum pH range i.e. 6-7 would result in hindering its availability in soil. In agreement with this, Chandna et al. [38] has also correlated the solubility and stability of these minerals with pH of the mature compost.
In the current study, fungal community dynamics was studied at five different incubation temperatures with different incubation times. In our study, it was observed that the mesophilic fungal load had increased not only with the passage of time but also with a rise in temperature whereas thermophilic fungal community underwent a decline even during thermophilic phase. Furthermore, as composting progressed towards the cooling stage, decrease in values of abiotic factors (pH, temperature, and moisture content) had resulted in high count of mesophilic fungi. In contrast to our study, Bhatia et al. [40] composted cattle manure and green vegetable waste under rotary drum composter and reported the decrease of fungal load with time during composting. The study indicates that this uniqueness in fungal population were influenced by the nutritional factors in feedstock and composting method used.
To investigate fungal ecology via culture-based method, a total of 50 isolates were preserved based on their incubation temperature, microscopic identification and cultural characteristics. But out of this, only 35 (70%) were categorized and their succession were explored. Remaining 30% of the isolates were excluded because of two reasons (a) repetitive preservation of same morphotype and (b) inability of their growth on sub-culturing into new vials. This sub-culturing inability into new vials was justified by López-González et al. [9] explaining the hindrance in isolation was occurred due to difference in nutrients, pH and specifically the competitive nature of microbes found in first culture media only. In the current study, species from phylum Ascomycota (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium sp. and Cladosporium sp), and phylum Zygomycota (Mucor and Rhizopus) were recovered through standard plate count. Reviewing the study done by Anastasia et al. [41], the results of fungal community are in agreement that recovered fungal isolates in our study are associated with the degradation of biowaste.
Later on, fungal strains that were isolated during composting were screened for their enzymatic capabilities of degrading complex substances into simpler ones. Since, the compost pile has been made from fruit waste, leaves, and saw dust, it became a rich source of pectin and cellulose, therefore, isolated fungal strains were investigated for pectinolytic and cellulolytic activities. Ten fungal isolates were found to be cellulase positive at 30 °C while only four fungal isolates were screened to be cellulase positive at 50 °C. On the other hand, only three fungal isolates were found to be pectinase producers at mesophilic and thermophilic temperatures. These findings are in conformity with the findings of other studies that reported the capability of genera Aspergillus and Penicillium for the cellulase and pectinase production [21,[46][47][48][49]. Overall, all lab designated isolate numbers (ADIF1, ADIB8 and ADIK2) of selected Aspergillus fumigatus were able to exhibit both cellulolytic and pectinolytic activity at thermophilic temperatures. Phutela et al. [50] has also reported the efficacy of A. fumigatus isolated from decomposing orange peel as a best producer of pectinase.
Culture-based analysis is not enough to give a true representation of microbial community in a rich environment like compost. It has also been reported that synthetic media has limited the species diversity and could only cultivate estimated < 1.5% of microbial diversity which shows biasness of culture-based results [20]. However, its significance should not be denied, and it must be coupled with the culture-independent techniques. Therefore, we opted DGGE and illumina sequencing technique that uses extracted DNA from microbial community of compost sample and enable us to have a better representation of diversified fungal community.
DGGE revealed that the fungal community is far more diverse than presented by culturing. The decrease in the number of bands from week 0 to week 4 suggesting that the fungal specie cannot survive higher temperatures [7]. It has been reported in several studies that when the temperature of the compost pile reaches > 40 °C, fungal growth was not apparent [3,52]. But when the temperature drops, the fungal population starts recolonizing the compost [2,3]. Haseen et al., [53] determined the relationship of physicochemical conditions and microbial population present in compost of municipal solid waste. According to them at ca. 60 °C, the count of yeasts and filamentous fungi decreased from 10 6 to 10 3 cells g −1 , this could be because of inability to grow and death. Although significant numbers of fungi survived and were able to recolonize the compost once the temperature dropped. Substrates availability, temperature and moisture content seemed to play a key role in designing the composition of fungal community, present at the respective stages of the composting process [51].
DGGE is an important technique but like other molecular techniques, it has limitations too. Several reports are published reporting the limitations of DGGE e.g., co-migration of bands could underestimate while heterogeneity of genes could overestimate the diversity. Use of universal primers for amplification of gene could results in prevalence and ignorance of predominant and minor constituents of community, respectively [55][56][57]. Cutting and sequencing of DGGE gel bands could enhance the resolution of DGGE, although the attempt failed in our case (data not shown).
Although DGGE is a sophisticated technique, but we cannot rely on one technique for in-depth study of microbial functional ecology that's why we also opted for Illumina sequencing. In matured compost sample, ITS amplicon based illumina Miseq platform revealed fungal diversity associated with three phyla including Zygomycota, Basidiomycota and Ascomycota and among phyla, Zygomycota was found as the most dominant one. Among Zygomycota, Mortierella sp. and Rhizomucor sp. were identified that are usually associated with decaying of the plant organic matters and appear in both mesophilic to thermophilic condition especially during composting of lignocellulosic materials [42]. Both species cause lignocellulose degradation and are mainly associated with hemicellulose and cellulose degradation [43]. However, most of Mortierella sp. and Rhizomucor sp. are usually termed as saprobe, ectomycorrhizal or plant pathogens that invade and take advantage to utilize soluble organic matters. Basidiomycota revealed as second dominant phylum, with Coprinopsis sp., Coprinellus canistri & Coprinus cordisporus. These species belong to class Agaricomycete which are considered as efficient ligninolytic fungi with potential extracellular enzyme system. Most of these fungi can easily be seen on decaying wood with large fruiting bodies. They cause degradation of lignin first, leaving behind cellulosic material that usually utilize later [44]. Based on abundance, it can be understood that there must have been a gradual shift in wood-rotting fungi replaced with opportunist fungi i.e., zygomycetes. Results indicated less abundance of Ascomycota which are generally involved in soft decay of wood and show comparatively less potential of wood degradation than basidiomycetes. They are mainly attributed to hemicellulose and cellulose degradation [45]. The lowest abundance of ascomycetes can be linked with appearance of zygomycetes as opportunists on mature compost that attacked cellulosic material with highest abundance and deprived ascomycetes from its utilization. In addition, Several studies using pyrosequencing, suggested that at thermophilic stage of composting process, there is selection of thermophilic and thermotolerant fungal population which decreases the fungal diversity at this stage [2,7,54]. In the study, Zafar et al. [20] incubated compost at 45 °C and 50 °C for 12 weeks and reported that, Emericella rugulosa, and Scytallidium thermophilum accounted for 81% and 88.9% of all sequences at 50 °C and 45 °C respectively.
Genus Aspergillus was the most recovered fungi via culturing but according to the DGGE and sequencing results it was sparsely present in compost. Similarly, in a previous study using 454 pyrosequencing, A.fumigatus and T.lanuginosus accounted for < 0.35% of the sequences, despite appearing to be the only organisms through conventional cultivation [7]. Culture based techniques depends upon the conditions of cultivation that favors the proliferation of fungi with less biomass from small spores number which is too small to be detected by PCR based methods [51]. That's why it is important to couple culture-based techniques with molecular methods.
Lastly, as far as, phytotoxicity of compost is concerned, GI value of 80% indicates absence of phytotoxicity in compost [58] and in our present study the fruit waste compost did not exhibit phytotoxicity in Vigna radiata seeds resulting in GI value of 83.3% as compared to cattle manure and chemical fertilizer. In the similar study, Choy et al. [59] performed the phytotoxicity of product obtained after cocomposting of horticulture waste with fruit peels, food waste and soybean residues separately and observed that GI value were better with the product co-composted with fruit peels i.e. 125%. This suggests that use of fruit peels in composting gives better maturity to compost and result in significant root growth of seeds.
All things considered; our study has concluded that fruit waste is most suitable biowaste for composting that produces enriched compost which could be used as an organic fertilizer. Our study has also revealed that primary combination of sawdust and fruit waste provided a balanced nutrient and good moisture content to carryout composting process efficiently in a sub-tropical environment within a period of 146 days. Moreover, the assessment of physicochemical parameters assured the maturity and quality of compost and its transition had influenced the fungal community shifts during composting process. The periodic growth of fungal population over time indicates that they are secondary degraders involved in the decomposition of recalcitrant substrates.