3.4 Characterization of microbial isolates
Microscopic investigation revealed that PM-4 colonies were glubose in shape and smooth walls with extremely uneven and asymmetrical conidia exterior and identified as A. niger. Gram staining of bacterial isolates displayed positive rods for BS2, CD2E, and BTA with the ability to form endospores. While CD6C was gram-negative rods. Bacterial strains BS2, CD2E, BTA, and CD6C were characterized on the molecular level (Table 1).
Table 1
Identification of bacterial strains based on 16S rRNA sequence
Isolates | 16S rRNA Gene | Accession No | Subject | Score | Identities |
| | | Length | Start | End | Coverage | Bit | E-Value | Match/Total | Pct% |
CD2E | B. altitudinis | NR_042337.1 | 1506 | 20 | 1506 | 98 | 2741 | 0.0 | 1487/ 1488 | 99 |
CD6C | P. aeruginosa | CP012001.1 | 6317050 | 696959 | 698425 | 0 | 2710 | 0.0 | 1467/1467 | 100 |
BS2 | B. velezensis | KY694464.1 | 1508 | 17 | 1506 | 98 | 2723 | 0.0 | 1485/1490 | 99 |
BTA | B. wiedmannii | KU198626.1 | 1540 | 17 | 1491 | 95 | 2652 | 0.0 | 1466/1479 | 99 |
Note: Results are the average of triplicates |
A phylogenetic tree was constructed showing percent similarity to the closest known bacterial strain. After 16S rRNA sequencing, strains were identified as B. wiedmannii BTA, B. altitudinis CD2E, B. velezensis BS2, and P. aeruginosa CD6C (Fig. 1). These selected isolates were used as bio-inoculant for composting PM to convert them into biofertilizers.
3.5 Hydrolytic enzyme production
3.5.1 Cellulase
In the present study, four microbial isolates were designated as effective cellulase manufacturers owing to the development of the zone. After two days of incubation solvent zone of cellulose was generated around the microbial isolate. The isolates B. altitudinis CD2E, B. velezensis BS2, B. wiedmannii BTA, and A. niger PM-4 were found to produce a hydrolytic zone by using CMC exclusively as a carbon source. The enzyme production curve of selected isolates with varying temperatures (20-50Co) (Fig S1) and pH was shown (Fig. S2).
The maximum CMCase activity of B. velezensis BS2 (7.0 ± 0.9 U/ml) was found at 35Co, however, the highest enzymatic activity of B. altitudinis CD2E (5.9 ± 0.2 U/ml) and B. wiedmannii BTA (9.0 ± 0.1 U/ml) were observed at the optimum temperature of 40Co. The maximum quantity of CMCase (18.2 ± 0.01U/ml) was attained when A. niger PM-4 was raised at 40oC (Naeem et al. 2021). The maximum CMCase activity of B. velezensis BS2 was found at initial pH 5 (6.5 ± 0.1), however, the highest enzyme activity of B. altitudinis CD2E (5.9 ± 0.2 U/ml) and B. wiedmannii BTA (9.1 ± 0.1 U/ml) were obtained with initial pH 6 of the medium. Meng et al. (2014) and Li et al. (2020) also informed similar findings for the enzymatic activities of Bacillus. Spp. The optimal cellulase enzyme production conditions of the B. velezensis strain were 35°C and pH 5.0. Sreena and Sebastian, (2018) measured the maximum cellulase production by B. subtilis strain (187.08 ± 2.8 U/ml) at 40°C. The highest enzyme activity of A. niger PM-4 originated with the primary pH 6 (19.0 ± 0.09) (Naeem et al. 2021). The outcome of temperature and pH deviation tendency of cellulase by A. niger PM-4 supports the findings of El-Hadi et al. (2014) and Sohail et al. (2009). Enzyme action was also perceptible at initial pH 7 (18.2 ± 0.01 U/ml). All microbial strains showed an increasing trend towards increasing temperature and pH, however, after reaching optimum temperature and pH, enzyme activity becomes reduced. Similarly, enzyme activity also increased gradually with an increase in incubation temperature from 20oC up to optimum temperature and after that slowly decline.
3.5.2 Xylanase
Among selected microbial strains, B. wiedmannii BTA and A. niger PM-4 showed positive xylanase activity on the substrate birchwood xylan. Numerous research findings describe the activity of A. niger to release xylanase, however, no research data is available for B. wiedmannii BTA for production and optimization of xylanolytic activity. Optimum enzyme activity for B. wiedmannii (15.1 ± 0.2) and A. niger PM-4 were achieved (24.7 ± 0.09) at 40 Co and 45Co respectively. The lowest quantity of enzyme (8.9 ± 0.1) was notable at 20Co. The production of enzymes rises with increasing temperature and subsequently receiving to the optimal temperature, and a steady decline is noticed. Though, the production of enzymes remains dynamic at a boosted temperature of 50Co (Fig. S3).
Optimum pH for both xylanolytic strains, B. wiedmannii BTA and A. niger PM-4 displayed its greatest release at pH 7.0. At higher pHs 8 and 9, the activity of the enzyme becomes very small (Fig. S4).
Previous research findings frequently reported alkali tolerant xylanase-producing Bacillus spp. work best at pH 8, however, few studies reported xylanase enzyme production by Bacillus strains at neutral pH. The current result findings agreed with Monisha et al. (2019) described optimum pH 7 for xylanase enzyme by enzymatic hydrolysis of beechwood xylan (Fig S4). A. niger PM-4 optimum temperature and pH range were outcomes of (Pal and Khanum, 2010; Uday et al. 2017). Several former investigations informed xylanase enzyme production by A. niger fluctuated from pH 3.0 to 9.0 (Bajaj et al. 2010).
3.5.3 α-amylase
In this study, B. velezensis BS2, B. wiedmannii BTA, and A. niger PM-4 were measured for the production of α-amylase. The optimal α-amylase production by the B. velezensis BS2 (4.8 ± 0.1 U/ml), B. wiedmannii BTA (1.95 ± 0.2 U/ml) strain was achieved at 35°C and 45°C. The temperature rise caused higher enzyme production up to the optimum temperature and after that gradual decline was observed. Mostly, α-amylase producing bacterial strains showed a 6–7 optimal pH range for enzyme production (Gupta et al. 2003). The highest production of amylase was noted at 30°C by A. niger (25 ± 1.1 U/ml) (Fig. S5).
The optimum pH for enzyme production was noted at pH 5.0 and 6.0 by B. velezensis BS2 (3.0 ± 0.2 U/ml) and B. wiedmannii BTA (1.7 ± 0.1 U/ml) respectively. Mushtaq et al. (2017) reported that the B. subtilis K-18 strain exhibited higher amylase production at pH 5. Less than and greater than pH 5, abridged amylase production (Fig. S6). Ullah et al. (2021) reported optimum activity by IR-8 B. wiedmannii strain at 45C0 temperature and pH 5.9. For optimum enzyme activities, favorable incubation temperatures ranged from 30°C to 50°C. B. wiedmannii can produce endophytic spores, which can bear a wide array of physical and chemical situations in terms of pH and temperature (Chen et al. 2018). The Lactobacillus LMG 18010 T strain depicted maximum extracellular alpha-amylase enzyme production at the optimal temperature of 55oC with pH 5.5. It was further determined that the amylase constancy was better in the 5 to 6 pH range (Aguilar et al. 2000). Mohamed et al. (2009) studied a comparable analysis, which showed the alpha-amylases enzyme stability up to 50°C and some of them at 40°C. The greatest amylase generation by A. niger was detected at pH 6 (24.5 ± 1.2 U/ml) and 7 (24.0 ± 1.1 U/ml) respectively. The outcomes for optimal temperature and pH by A. niger PM-4 followed the findings of Abdullah et al. (2014) and Rose´s and Guerra, (2009) and the tendency in deviation was conferring Wang et al. (2016).
3.5.4 Pectinase
Pectinolytic enzyme activity was noted by B. wiedmannii BTA and A. niger PM-4 by hydrolyzing pectin from apples. This study first time used B. wiedmannii BTA for the production and optimization of the pectinase enzyme. The highest enzyme activity by B. wiedmannii BTA (5.5 ± 1.1) was achieved at a temperature of 35Co. The pectinase activity increased as the incubation temperature raised from 20 to 35°C and reached its highest at 35°C, and with further rising temperature, enzyme activity remain stable up to 50Co. Consequently, the optimal temperature for pectinase generation is 35°C. The A. niger PM-4 showed maximum pectinase activity at 30oC (25.0 ± 1.9 U/ml) and it continued constant up to 40Co and afterward, slowly decline (Fig. S7). The highest enzyme activity by B. wiedmannii BTA (5.5 ± 1.1) was achieved at pH 7. Furthermore, at the acidic pH, the pectinase enzyme is more stable and active than at the alkaline pH. It is noted that microbes had thermal stability (30–50°C) and higher pectinase activity at pH 4.0–7.0 (Fig. S8). The findings are following the pectinolytic activity of Bacillus sp. ZJ1407. The optimum pH and temperature were 5.0 and 37°C respectively (Yu et al. 2018).
Initial pH 6 released more pectinase enzyme by A. niger PM-4 (Fig. S8). As the pH level raised from 7, a decline in pectinolytic activity was noted (Naeem et al. 2021). Several other researchers noted the maximum enzyme release (117.1 ± 3.4 U/ml) by A. niger at a 5.5 pH and a temperature of 30 C0 (Ahmed et al. 2016). Regulating pH 5.5 during microbial generation produced a 109.63 U/mL pectinase, which is better than uncontrolled pH (El Enshasy et al. 2018).
It has been clear from the findings that selected microbial strains are temperate thermophiles with 20, 30–40 and 50°C, minimum, optimum and maximum temperature for cultivation respectively. Microbes do their best performance at acidic to neutral pH. pH 8–9, 5–6 and 7 are noted as minimal, optimal and maximal for enzyme activity. The temperature directly above 55Co suppress enzyme activity by A. niger PM-4, however, bacterial enzyme activity work even at higher temperature (Naeem et al. 2021). The results findings for all hydrolytic enzyme production noted that A. niger PM-4 had the greatest potential of releasing a higher amount of enzyme compared to bacterial isolates, however, bacterial isolates can tolerate a higher temperature range.
Plant growth stimulating properties
3.6.1 Biological nitrogen fixation
The treatment of nitrogen-fixing bacteria as bio-fertilizers has appeared as a sustainable way to augment the productivity and growth of crops (Gupta et al. 2020). Microbial strains B. altitudinous CD2E and P. aeruginosa CD6C were found efficient in fixing atmospheric nitrogen. The positive activity of microbial strains changes the media color to blue indicating efficiency for nitrogen fixation. It is the indication of secretion and accumulation of ammonia in their growing environment which changes the pH of the media and ultimately leads to color change. The finding was per Latt et al. (2018) who described that a change in color of indicator dye BTB suggests excretion of ammonia. Microbes produce ammonia through mineralizing nitrogen from urea, while releasing CO2 and NH3 (Rodrigues et al. 2016). Strains of B. altitudinis and Pseudomonas spp. formed relations with plants by fixing atmospheric nitrogen (Habibi et al. 2014). Gupta et al. (2013) characterize the diazotrophic nature of P. aeruginosa even after sub-culturing for several generations and showed an efficient reduction of acetylene through the “acetylene reduction assay”, which is produced by the activity the of the nitrogenase enzyme.
3.6.2 Nutrients solubilization
The addition of mineral P fertilizer to the rhizosphere enhanced crop yield and is extensively practiced all over the world in agriculture. However, some limitations exist like mineral P is rapidly absorbed in the soil and becomes unavailable. The application of PSB is a substitute for the arising issue. The current research reported the capability of A. niger PM-4 (Naeem et al. 2021)d aeruginosa CD6C to solubilize TCP in solid and liquid media. The transparent zone formation around microbial growth on NBRIP agar media is a suggestion that the isolate had P solubilizing capability. Additionally, strain efficiency in NBRIP broth showed the highest P solubilization activity of A. niger PM-4 and P. aeruginosa CD6C was found on the 8th day of incubation 389 ± 1.9 and 110 ± 1.1 µg/ml. A gradual increase in P solubilization and a significant drop in pH of the media were also observed. The findings agree with the reported outcomes by Galeano et al. (2021) who confirmed a steady rise in soluble phosphate contents by fungal strains in specified liquid cultures. pH Dropping during P mineralization by microbes is in complete accordance with (Bakri, 2019) stated slow alteration in pH throughout a week of incubation. Several mechanisms are involved in the P mobilization by rhizospheric microbes, like the release of organic acids, reduced pH, and formation of phytases and phosphatases. Several other scientists described A. niger strains as producing a substantial quantity of acid phosphatase (Nahas, 2015). A. niger PM-4 was found more efficient in producing a higher amount of P as compared to P. aeruginosa CD6C. Former studies by scientists revealed that fungal isolates are higher P solubilizers in contrast to bacteria (Bakri, 2019; Wang et al. 2018).
P. aeruginosa CD6C and A. niger PM-4 (Naeem et al. 2021) were isolated as potential zinc mobilizers and noted to solubilize insoluble ZnO and at the 8th day of incubation releases 55 ± 1.2 µg/ml and 155 µg/ml Zn correspondingly. A significant fall in pH 4.9 and 5.2 for A. niger PM-4 and P. aeruginosa CD6C with initial pH of 7 was also recorded. Several species of Aspergillus and Pseudomonas have broadly studied their relationship with plant growth performance (Anuradha et al. 2015) by mobilizing Zn as well as P solubilization.
The current study determines the potassium mobilizing activity of P. aeruginosa CD6C by the creation of the transparent zone on Aleksandrov culture media containing an insoluble form of K as feldspar (Naeem et al. 2022). However, very low potassium solubilizing activity was recorded after eight days of incubation (27.1 ± 1.1 µg/ml) with a significant drop in pH and reduced the pH to 6.1 from the initial pH of 7.0 of the medium.
3.6.3 IAA production
Plant growth hormone such as IAA generation is widespread among members of bacterial genera including Rhizobium, Pseudomonas, Agrobacterium, Bacillus, and Enterobacter (Etesami et al. 2015). P. aeruginosa CD6C was found to produce the growth hormone IAA in both qualitative and quantitative analysis. A Cherry red color appears by adding a few drops of Kovac’s reagent and light pink with the addition of 1 ml of Salkowski's reagent respectively. Quantitative assessment through HPLC measured 2.81 µg/ml of IAA. Ali et al. (2020) evaluated different strains of pseudomonas for IAA production and found that Pseudomonas strains, LB1, and FB5 produced 8.8 µg m/l, and 9 µg m/l the highest amount of IAA respectively.
3.6.4 Biocontrol agent
A total of two bacterial isolates B. velezensis BS2 and P. aeruginosa CD6C displayed biocontrol activity against potential plant pathogens. The plate culture assay revealed that the antagonist bacterial strain inhibits the mycelial growth of pathogenic fungal strain. Pseudomonas and Bacillus species are extensively renowned as biocontrol agents for their capacity to activate efficient struggle by the formation of endospore and antagonistic effects that make them useful in the formulation of products with the accumulation of different mechanisms of action (Wang et al. 2020). In Korea, 14 microbial fungicides products contain Bacillus species (Rabbee et al. 2019). B. velezensis, have been extensively used in bio-formulated commercial products to prevent potential pathogens. Planta’s evaluation exhibited that arugula plants applied with Bacillus and Pseudomonas strains reported maximum percentage subsistence and disease control (Ali et al. 2020).
3.6.5 Compatibility between microbial isolates
Compatibility evaluation using a cross-streak test on nutritional agar medium indicated that the four bacterial isolates selected were compatible with each other and did not inhibit each other's effect. However, P. aeruginosa CD6C and B. velezensis BS2 suppress the growth of A. niger PM-4 due to their antagonistic activity. Purified metabolites of Pseudomonas and Bacillus strains exhibit significant antifungal activity (Ali et al. 2020).
3.8 Variation in physicochemical characteristics
3.8.1 Temperature variation
The temperature of the substrate subjected to composting identifies the volume of biological breakdown and the suitable period for the development of appropriate environments assisting microbial breakdown.
The T2 (PM+A. niger) and T3 (PM+ consortium) heaps temperature raised promptly, recorded in thermophilic stage at day 5, though, attained its highest level at day 10 which was sustained at a comparatively elevated level up to the day 20. The temperature slowly weakened at the completion stage of the thermophilic period and headed towards a stable and developmental stage of composting. A relatively greater temperature was detected in the heap comprising bacterial consortium T3 (PM+ consortium) than any other treatment owing to the microbial activity and faster degradation rate of the substrate as compared to T2 (PM+A. niger) and T1 (Control). The control treatment thermophilic stage starts at day15 due to slow rising temperature and becomes unsuccessful to achieve temperature throughout the composting period as reached by inoculated heaps (Fig. 2a). Former researchers noted the optimum temperature of 50°C–60°C for effective breakdown, which is attained on the 25th day in the control heap (Awasthi et al. 2014). The slower rise of temperature in the control heap and shorter thermophilic stages indicated the lack of efficient initiator bio-inoculant overdue decay. The bio-inoculant inclusion altered the rate of the rising temperature of the composted material and subsequent heat generation (Meng et al. 2018). Rising temperature in heap containing bacterial consortium accelerates the breakdown of substances, therefore, exhaustion of nutrients, due to which the temperature gradually decreases and enters the cooling phase for maturation (Yu et al. 2020). The comparatively higher temperature of the T3 (PM+ consortium) compared to T2 (PM+A. niger) suggested that the increase may be due to the consortium influence which satisfies the findings of Barthod et al. (2018).
3.8.2 pH variation
Owing to the fermentative metabolism, the pH of all treatments first lowers which resulted in the production of a higher amount of organic acids. Afterward, it enhanced (Fig. 2b) comparable to the findings described by Awasthi et al. (2014). During the first five days, the pH of heaps T2(PM+A. niger) and T3 (PM+ consortium) reduced and then rise at the completion stage of composting, whereas control heap pH was reduced up to day 20 and afterward lightly increased (Fig. 2b). The low pH at the initial stage of composting is owed to the generation of organic acids by microbial action. Nakasaki et al. (2005) describe that nitrogen-fixing microbes transform NH3 into NO2 and subsequently nitrate under adequate oxygen contents. During the process of nitrification, H + ions are released which lowers the environment's pH. The progressive pH increase is attributed to the generation of ammonical nitrogen by the disintegration of compound proteins (Awasthi et al. 2014). The aerobic environment supports organic N mineralization into ammonia through ammonification, and the pH value of the heap increase (Wong et al. 2001). A relatively slow level of control heap pH might be due to the low level of microbial action which leads to slow biodegradation of the substrate.
3.8.3 EC variation
EC represents fertilizer salt content, mainly describing the biofertilizer characteristics. The heaps T1(control), T2(PM+ A. niger), and T3(PM+ consortium) EC values were 1.55 ± 0.01, 1.52 ± 0.04, and 1.51 ± 0.01 mScm − 1 respectively, and progressively raised as the composting process progress (Fig. 2c). The primary EC rise is owed to the formation of simple compounds and mineral ions (Awasthi et al. 2014). The EC values were higher for treatments inoculated with microbes compared to control. No differences were observed between treatments inoculated with A. niger and bacterial consortium, however, bacterial consortium inoculation raised the EC value to 2.5 mS/cm and it was achieved on day 25 and remained constant on day 30. EC > 4 mS cm − 1 specifies the occurrence of more soluble nutrients and will badly affect plant growth rate e.g, slow rate of germination and withering, etc. Low EC fertilizer can be directly applied to the crops; however, greater EC fertilizer needs to be incorporated with any other media or soil. At the completion of composting, all treatments showed a higher EC limit, however, below the toxic category.
3.8.4 TOC%, TKN, and C/N variation
Data in Fig. 3a exposes a steady decline in TOC during composting. However, higher TOC reduction was obtained after a month in T2 (PM+A. niger) and T3 (PM + consortium). The low TOC of heaps inoculated with microbes represents a prompt breakdown of organic carbon. Fungal and bacterial strains were found to release enzymes accountable for the rapid breakdown of lignocellulosic waste (Alberts et al. 2009; Esawy et al. 2013).
The preliminary TOC contents were 60.9%. Though this percentage decayed sharply and it was observed that after one week T1 (control), T2 (PM+A. niger), and T3 (PM+ consortium) reduced the TOC% by 11.2%, 17.1% and 18.8% respectively. At completion, T3 (PM+ consortium) reduced higher TOC followed by T2 (PM+A. niger) and T1 (control). T3 (PM+ consortium) was found to be 6.7% more efficient in reducing TOC as compared to T2 (PM+A. niger). Voběrková et al. (2017) describe micro-consortiums as efficient in reducing TOC as compared to individual microbial strains. The efficiency of waste decomposition and compost maturation depends on the kind of microbe used for inoculation.
During the composting period, the TKN of the inoculated and control heaps fluctuate (Fig. 3b). During 1st week low TKN was noted in all treatments proposing an enhanced degradation of proteinous substances that might have reduced TKN, and generated ammoniacal nitrogen (Awasthi et al. 2014; Gou al, 2017; Yang et al. 2018). Finally, TKN content in all heaps was continuously enhanced till completion, however, improved TKN was obtained with inoculated treatments. T3 (PM+consortium) enhanced 8.25% TKN as compared to T2 (PM+A. niger) (Figure. 4.31b). The elevated level of TKN at the end was principally due to the concentration result. Additional constraints may include augmented nitrifying microbial action (Nakasaki et al. 2013). Even in the absence of external nitrogen supplies, Gou et al. (2017) reported enhanced TKN.
The initial C/N ratio of heaps was 40.6 and finally reduced to 21.4, 17.4, and 16 after a month for T1 (control), T2 (PM+A. niger) and T3 (PM+ consortium) respectively. T3 (PM+ consortium) reduced 7% more C/N as compared to T2 (PM+A. niger) (Fig. 3c). Inoculated heap containing a lower C/N ratio satisfied the standard of mature compost as compared to control as the higher C/N would cause nitrogen to volatize by reducing the nitrogen content and lower C/N would generate an appreciable amount of soluble salt thus making the soil enhance the plant growth (Raut et al. 2008). The TOC and C/N ratio of the matured compost should be less than 40% and 20 respectively (Raut et al. 2008; Wei et al. 2019). In comparison to control, biofertilizer inoculated with bacterial consortium found its maturity at the 21st day (C/N less than 20), however, A. niger PM-4 inoculated biofertilizer become mature after a month. This result declares that PM inoculated with efficient microbes could be successfully converted into compost during a month, however, without inoculation, it results in incomplete composting. Heidarzadeh and Amani, (2019) reported that inoculation of microbial strains can increase product quality and reduce the time of the process and operating costs.
3.8.5 TP and TK variation
The initial TP in the T1 (control), T2 (PM+A. niger), and T3 (PM+ consortium) heaps were 1.1%, 1.12% and 1.2% (Fig. 4a). As the process headed, these quantities raised expressively by 1.3%, 1.42% and 1.42% for control and inoculated heaps till the completion of composting. As compared to the control, the inoculated heaps contained 13.6% more TP. A similar increasing trend was also observed in TK contents of inoculated and control heaps (Fig. 4b). At completion, the TK contents were 0.74%, 0.83%, and 1.0% in the T1 (control), T2 (PM+A. niger) and T3 (PM+ consortium) heaps correspondingly. T2 (PM+A. niger) noted 8.1% higher TK as compared to control, however, T3 (PM+ consortium) enhanced 20.3% more TK as compared to T2 (PM+A. niger).
In control heaps, TP and TK contents were slowly added, however, inoculated heaps showed more pronounced results. Several research outcomes noted improved TP and TK contents throughout composing even in the absence of added mineral sources (Awasthi et al. 2014; Li et al. 2019). Moreover, Li et al. (2019) also noted an enhanced TP and TK contents throughout composting. These results observed that enhanced organic matter breakdown over time increases the quantity of these nutrients, moreover, microbes also transform nutrients into available forms
3.8.6 CEC variation
Another imperative constraint for mature compost is CEC (Karak et al. 2013). The initial and final CEC values for all treatments are listed in Table 4. At the end of the composting period, higher changes were detected in the inoculated treatment T2 (PM+A. niger), and T3 (PM+ consortium), though, the control treatment noted a minor increment in CEC.
Table 4
Variation in CEC throughout composting
Sr No | Treatments | Initial CEC | Final CEC |
1 | T1 (control) | 242.6 ± 31.2 | 314.2 ± 43.2 |
2 | T2 (PM+A. niger) | 251.4 ± 30.1 | 623.4 ± 31.1 |
3 | T3 (PM+ consortium) | 217.1 ± 28.1 | 691.4 + 48.1 |
Note: Results are the mean of triplicates |
The higher CEC levels were generated due to the formation of phenolic and carboxyl functional groups. For all three treatments, the mean CEC ratio increased as decomposition time increased. The most likely cause of rapid PM mineralization is oxidizing radicals and molecules which direct to enhanced CEC values (Sahu et al. 2019). Table 4 reveals that the mean CEC value in the T3 (PM+ consortium) treatment was substantially greater than the T2 (PM+A. niger) and T1 (PM+A. niger) treatments (control). The findings back up the theory that microbes accelerate substrate degradation, resulting in a higher CEC value (Sahu et al. 2019).
3.8.7 SEM observation
Figure 5 presented the SEM photographs of PM during 30 days of composting. The photographs revealed that the substrate inoculated with microorganisms was expressively eroded during 30 days of composting. Bacteria, as well as fungi, were seen on the film surface. Fungal mycelium growth was observed on the substrate surface (Fig. 5b, 5c, 5d). Figure 5e, 5f, 5g demonstrate the large number of microbes rising on the substrate surface. Surface erosion, patterns, and crashes after microbial inoculation were also realized (Fig. 5d, 5g). The surface of the control substrate was a little eroded and remained smooth. SEM photographs of PM showed that before composting substrate is thick and massive (Fig. 5a) which leads to very little erosion or breakdown of the substrate surface in the control treatment (Fig. 5h, 5i, 5j), however, SEM analysis of PM inoculated with A. niger (Fig. 5b, 5c, 5d) and bacterial consortium (Fig. 5e, 5f, 5g) revealed prominent growth of fungal mycelium and bacterial growth with loose and porous materials respectively suitable as biofertilizer. Mei et al. (2020) showed a significant difference by equating SEM photographs of straw fermentation with and without B. amyloliquefaciens. The results displayed the remnants of lignin cultured without B. amyloliquefaciens SL-7 were higher in size as compared to the strain B. amyloliquefaciens SL-7
3.8.8 Determination of phytotoxicity
The GI of T1 (control) (75.5%), T2 (PM+A. niger) (96.5%) and T3 (PM+consortium) (97%) were recorded after a month. The GI > 80 at the completion of composting duration depicting mature and non-phytotoxic biofertilizer, however, the control heap GI T1 (control) (75.5%) represent an unstable, immature, and phytotoxic product. Microbial inclusion enhanced phytotoxic element exclusion and boosted GI (Wan et al. 2020).