5.1 Changes in Physicochemical parameters:
Temperature of compost pile (Surface and Centre) and environment was monitored throughout the composting process that started on zero day and ended on 146th day. Changes in temperature profile were observed in Figure 1, depicted that as compared to the ambient temperature. 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 easily-biodegradable organic compounds . With progressive degradation, temperature of the compost pile gradually dropped and thereafter remained consistent with time to ambient temperature. As shown in Figure 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 , while water in the center remain trapped.
The variation in pH and EC throughout the composting process is shown in Figure 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 that includes calcium, magnesium and phosphorus 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 Calcium and Magnesium a significant increase (P<0.05) was observed in samples collected from initial and final stages of composting. Whereas Phosphorus content was found 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 with a drop in C content and C:N. The same period showed sharp rise in P and other macronutrients. 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.
5.2 Changes in Fungal Community during Composting via Culture-based Technique:
The count of viable fungi at different time points during composting showed an increasing trend at mesophilic temperatures (Figure 4, P<0.05). In the beginning, fungal count at 25 ºC was 5x106 ( + 1 x105) CFUg-1that significantly increased (P<0.05) to 3.76 x107 ( + 2.5 x106) CFUg-1in second week of composting and it ended at 1.5 x107 ( + 1.7 x107) CFUg-1 with several fluctuations in count. Similarly, at 37 ºC, initial count was 2.0 x106 ( + 2.5 x105) CFUg-1 that substantially increased to 4 x107( + 1 x106) with time (day 15, P<0.05). In contrast, thermophilic temperatures (45 °C, 50 °C and 55 °C) showed variations in their trend with the rise in temperature. At 45 °C, the fungal count was 1.46 x106 ( + 3.05 x105) CFUg-1 that significantly increased during thermophilic phase and reached to 5.3 x107 ( + 3.05 x106) CFUg-1 and ended at 7.6 x107 ( + 5.7 x106) CFUg-1 conferring an overall increasing trend. However, at 50 °C and 55 °C, the fungal count had significantly declined from 2.13 x106 ( +1.5 x105) to 1.4 x105 ( + 3.0 x104) CFUg-1 and from 1.8 x106 ( + 8.1 x105) to 7.0 x104 ( + 1.0 x103) CFUg-1 respectively, with the increase of pile temperature in the first week of composting. It was found that variation in fungal viable load existed throughout the composting period of 146 days. This variation was temperature- related which means the fluctuation in fungal count was not only observed within the temperature at different time points of composting process but also among incubated temperatures.
5.3 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 Figure 5. The findings show that Yeasts predominate in the early stages of composting at mesophilic temperatures due to presence of low pH and high moisture content. Whereas, on the later stages at mesophilic temperatures (25°C and 37°C) the phenotypic diversity of the fungal population was observed. At 25 °C, yellow powdery colonies with a white periphery and some black spores predominated, as did white cottony small-sized colonies. Similarly, at 37°C, white mycelial medium-sized colonies that matured to a greyish color, extremely dense black sporulating colonies, and gray powdery colonies were found to be similarly dominant morphotypes. However, no such diversity among fungal morphotypes was observed at thermophilic temperatures (45°C, 50°C, and 55°C). At 45°C, 50°C, and 55°C, the major morphotypes were green centered colonies with white periphery, white cottony colonies that turned brown over time, and white mycelial cottony colonies that swarmed over the entire growth medium on plate.
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. , this grouping and probable identification of isolates was done based on culture and microscopic morphology. Among those genera, Aspergillus spp. have dominated for the entire 146-day composting period (Figure 6). This demonstrated the fungal community's active participation in fruit waste degradation through composting.
5.4 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 (Figure 7). As the temperature goes down and compost gets mature, the diversity of fungal community increases. 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 (Figure 8 & 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. Band 1 only appeared in the week 4 and week 8 when temperature of the compost surface was around 40-50 °C. Band 2 corresponding 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. Band4 corresponds to Rhizopus oryzae which was frequently recovered in culture-based technique but appeared in low abundance through DGGE (Figure 7).
Another interesting observation revealed through DGGE 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 culturing.
5.5 Biodiversity of fungal communities associated with UTBC 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 know the complexity of sample, alpha diversity was applied through indices such as observed species (Figure 2 supplementary file) Chao1, Shannon- index and Simpson (Table-2 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 3 phyla including Zygomycota, Basidiomycota and Ascomycota and among phyla, Zygomycota was found as the most dominant one with relative abundance 52.16 % (Figure 10). On species level, Mortierella sp. and Rhizomucor sp. were identified. Basidiomycota revealed as the second dominant phyla with relative abundance 46.04 %, identified species were Coprinopsis sp., Coprinellus canistri & 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 Figure 11. The identified illumina sequences were submitted to NCBI GenBank for accession numbers as mentioned in (Table 3 Supplementary file)
5.6 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 cellulose, and therefore fungal strains were investigated for pectinolytic and cellulolytic activities.
Carboxymethyl cellulose 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, A. fumigatus (ADIF1, ADIB8 and ADIK2) grown and exhibited cellulolytic activity at thermophilic temperature. The highest cellulolytic activity was shown by ADIF1 (A. fumigatus) at 50 ºC, 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, A. flavus and Penicillium sp. were observed as pectinase producers at 30 ºC and A. fumigatus exhibited pectinolytic activity at 50 ºC.
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 chosen for identification through amplification of ITS gene (Table 1 supplementary file). DNA of Cladosporium sp. could not be recovered that is why its molecular identification was not performed. Overall, the results suggest 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.
5.7 Phytotoxicity of compost:
Germination Index (GI) was calculated by combining the measure of relative seed germination and relative root elongation. From the results obtained, GI of compost was 83.3 % hence indicating its maturity and absence of phytotoxic compounds (Table 3), which is higher chemical fertilizer (P>0.01) and animal manure.