Isolation, Characterization and Antifungal Behavior of Humic Acid and Fulvic Acid Fractions from Biowaste Derived Vermiproducts

Isolation, Characterization and Antifungal Behavior of Humic Acid and Fulvic Acid Fractions from Biowaste Derived Vermiproducts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, Characterization and Antifungal Behavior of Humic Acid and Fulvic Acid Fractions from Biowaste Derived Vermiproducts K. Vanimuthu, K. Kavitha, J. Arockia John Paul, P. Kumar, S. Gowrishankar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4221685/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Humic substances are widespread in the world and are formed from the plant and animal leftovers in the habitat when they are decomposed. The particular stuffs include three major combinations like humin, humic and fulvic acid. There are absolutely some reports available regarding the fungicidal activity of humic constituents. The objective of this study was humic materials determination and isolation from organic wastes and vermicompost deroved using the earthworm Perionyx excavatus for use to strength about the antifungal activity against the human fungal pathogen Candida species. Characterization of the fulvic and humic substances were done by various methods, including, UV-Vis spectroscopy, Fourier transform infrared, scanning electron microscopy and x-ray diffraction. According to the characterization results, it was observed that humic acid has a structure of honeycomb with fragments and flakes attached to the surface on it. The fractions also have a distinctive physical appearance of lumpy and close-grained. SEM and XRD analysis revealed that certain crystal forms occur in all humic and fulvic acid samples. The absorbances of peak between 250–500 nm have attributed significance nature of the humic acid. Further, total hydroxyls, amines, alkyl, alcoholic contents and acidic functional groups were determined. The growth of fungal Candida species was mainly affected by humic and fulvic acid and can reduce the usage of chemical drug that has several side effects. The earthworm plays major role in breakdown of organic substances to form humic substances, which is present in the vermicompost, vermicast and vermiwash. So, the approach of humic and fulvic acid taken from the vermiproduct is used to treat against the human fungal pathogen Candida species. Humic acid Fulvic acid Characterization Candida Perionyx excavatus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Microbial or chemical humiliation of plants and animal matter are the main sources to form humic acid substances (Myneni et al., 1999 ). Humic acids are insoluble in acidic whereas soluble in high pH conditions in an alkaline solution and are dark brown or black in colour (Tan, 2014 ). The process of forming humus is known as humification that forms through the organic matter decomposition. The presence of major acidic functional groups, specifically carboxylic and phenolic groups and /C, H/C and O/C atomic ratios are primarily determines the humus (Watanabe et al., 2009 ). From several resources, including coal, lignite, farmyard manure, vermicompost, etc., can be utilized for the extraction of humic acid. Hydrophilic groups in the humic acid encourage hydration and hence increase the soil’s capacity of retaining water. In soil and compost, it is the most efficient as well as the main component of humic substances. Vermicomposting involves employing earthworms with their flora that resides inside the digestive systems of them to convert new organic matter into stable organic matter (Atiyeh et al., 2002 ; Campitelli and Ceppi, 2008 ; Dores-Silva et al., 2013 ; Nogales et al., 2005 ). The humic acid (HA) and fulvic acid (FA) have physical and chemical characteristics extracted from various sites are dependent on large number of aspects smilarly the value of the original material. Humification process is a slow and progressive that can take thousands of years to complete in nature which leads to produce humic substances with various structural characteristics (Adani et al., 2006 ; Mignone et al., 2012 ). The use of decomposed materials is one of the most popular methods in organic farming. The particular substances bring about extracts of humic liquid that are usually composed of proteins, humic materials, and amino acids also contain variety of nutrients such as micro and macro nutrients. Vermicompost made from chicken litter, bovine manure, sewage sludge, urban trash and other renewable and natural sources are considered as the main sources to obtain liquid humic extracts (Warman and AngLopez, 2010 ). Humic acids provide essential nutrients to plants through the regulation of carbon cycle and release of nutrients from compressed soil (Kalsom et al., 2006 ; Stevenson, 1995 ). The oxidation-related damage can be inhibited or limited by performing the alkaline solution extraction under nitrogen environment, but the extraction process is still too harsh. The yield of humic acids is considerably lower with mild extractants including, cation exchange resins and sodium pyrophosphate when compared with alkaline solutions at the same extraction period. The solubility also varies in different extractants may be caused by variations in the macromolecules chemical structures or by organic macromolecules produced physically that interact to mineral fractions via cation bridges (Stevenson, 1995 ). The accurate information regarding the varied properties of humic substances and their fractions provided by combining multiple extraction techniques and fractionation processes (Nifant’eva et al., 1994 ). The maximum profit of HA extracted in Bintulu Sarawak from rehabitated forest, could be acquired in a period of 4 h of extraction were revealed (Hanisah et al., 2008 ). In the study, the discernible interaction between the extraction and the fractionation period was not reported. This is due to the fact that fractionation following acidification had no impact on the HA yield from the soils. However, the HA had to be precipitated from the restored forest soils for about 4 h is required for the purification using distilled water. Recent studies have shown that organic amendment can help prevent plant illnesses brought on by a variety of soil-borne phytopathogenic microbes, including fungus of different genera (Hoitink and Fahy, 1986 ). In that some investigations explained humic acids are suppressing of pathogen but they are reasonable for practice as biofertilizers in liquid form furthermore have been used against plant pathogen (McQuilken et al., 1994 ). The information regarding the fungicidal properties of humic acid, which is a component of vermiproducts like vermicompost, vermicast, and vermiwash is not available much in the literature. Therefore, the aim of the work is to isolate, characterize and to find out the antifungal activities of HA and FA in the vermiproducts (vermicompost, vermicast and vermiwash produced from biowastes) using alkaline precipitation method recommended by IHSS (International Humic Substances Standard). The characterization and purification of humic and fulvic acid done by SEM, FTIR, XRD and UV is useful for the preparation of HA and FA solutions in the precise concentrations needed for the treatment of the human pathogenic Candida species. Materials and methods Vermicompost unit construction Vermicomposting beds were constructed with the length of 10 m 3 , width of 8 and height of 3 m 3 in the shade place. The vermitech patterns were used to study the construction of vermicomposting units (Ismail, 2005 ). Earthworm culturing concrete tanks were set up of 150×100×60 cm 3 . To provide efficient water drainage, the concrete modules contained drainage holes (2 × 2 cm 2 ) were used. Preparation of vermiproducts To get worm-bed, four beds were used and roofs were placed over each bed to shield the vermicomposting beds from the sun light and rain. The 1 week aged cow dung and mixed biowastes (sugarcane waste, leaf litter, coconut husk) in 1:1 (v/v) was given as substrates for the earthworms ( Perionyx excavatus ) in each vermibed. The vermicompost bed was set to 80% vermiwash drainage and collection system for collecting vermiwash (Stevenson, 1995 ). Extraction of humic and fulvic acid About 10 g of air-dried vermiproduct samples, including, vermicompost, vermicast, vermiwash was deliberated in to 250 ml conical flask with 50 ml of 0.1 N sodium hydroxide and kept undisturbed for 24 h. After centrifugation, supernatant solution with dark colour was collected and the extraction process of humic acid was done repeatedly using 50 ml of extractant each time. Supernatants were combined in a conical flask, and the coloured solution was kept for 15 minutes in a centrifuge at 15,000 rpm to remove all the fine debris. In a 250 ml beaker, the coloured supernatant was collected after the clay was discarded. The pH electrode was immersed in the collected dark coloured supernatant solution and slowly 2 N HCl was added until it reaches pH of 2 to precipitate the humic acids. After giving it a thorough stir, it was let to retain for 24 h at the room temperature. Coagulate (HA), and supernatant (FA) were separately collected and on a hot air oven, the collected coagulate was dried. Spectroscopic characterization of humic and fulvic acids Acidic functional groups, including, phenolic (-OH) and carboxylic (-COOH) groups of HA were determined (Inbar et al., 1990 ). In a Thermo Nicolet 380 Fourier transfer IR spectrometer, Fourier Transform Infrared (FTIR) spectra was recorded from 4000 to 400 cm − 1 in KBr pellets which contained humic acids (0.50 mg) along with dry KBr of 200 mg (Schnitzer and Preston, 1986 ). FTIR spectra are regarded as HA and FA fingerprints for vermicompost, vermicast, and vermiwash. FTIR spectra results are evident that HA and FA isolated from vermiproducts contain distinctive functional groups and bonds. SEM analysis of humic and fulvic acid Extracted, dried HA and FA were maintained at 40 ºC and sent to USIC at Alagappa University for SEM observation. Instead of using acid, the samples of vermiproducts like vermicompost, vermicast, vermiwash (0.2 g) were produced for qualitative and quantitative analysis and examined using a SEM (Quanta FEG250-High Resolution) for their microstructure. E4/E6 ratio HA and FA were evaluated for their level of humification and aromaticity using E4/E6 ratio. At 10 ml of 1×10 − 2 M NaHCO 3 solution, a well-known quantity of the samples were taken and dissolved. The ratio and absorbance were recorded using UV-Vis NIR spectrophotometer at wavelength of 190 and 1100 nm. X-ray diffraction method X PERT PRO powder X-Ray Diffractrometer (XRD) system was used for the analysis of XRD patterns in the vermiproduct samples (Bhat et al., 2014 ). Crystalline structures present in the samples were analysed with software. Fungicidal activity against human pathogen Using mycelium growth assays, the in vitro fungicidal activity of HA and FA against Candida species were evaluated (Zhao et al., 2011 ). After dissolved in DMSO, HA and FA were diluted to 50 mg/L concentration in water that contained an emulsifier with 200 ug/mL. After that, 1 mL of test solution was put on sterile Petri plates with a diameter of 9 cm and finally 9 mL of agar culture media was added. The controls were maintained in 9 ml of agar culture media with samples lacking HA and FA. The inocula with a diameter of 4 mm was taken from the borders of energetically growing colonies and placed in the middle of the plates. Finally, zone of inhibition was measured by diameter after 72 h of incubation at 25°C. The plates were maintained in triplicates and the efficiency of inhibition of the HA and FA was calculated in terms of their inhibition percent (%). Strains and culture conditions In this present study, antifungal efficacy of HA and FA extracted from vermiproducts (vermicompost, vermicast, and vermiwash) was evaluated against three clinically important Candida species viz., Candida albicans (ATCC 10231; purchased from HiMedia, India), Candida glabrata (MTCC 3019; procured from CSIR-IMTECH, Chandigarh, India) and Candida tropicalis (MTCC 184; obtained from CSIR-IMTECH, Chandigarh, India). Strains were maintained in YEPD agar plate, from which, a loop-full of colony was used to inoculate YEPD broth at the temperature of 37°C for overnight in rotary shaker with 200 rpm agitation. To perform the agar well diffusion assay, 1 × 10 6 CFU/mL (0.1 OD) cells were used to swab on YEPD agar plates. Antifungal susceptibility assays Agar well diffusion technique previously designated by (Gonelimali et al., 2018 ) with required modifications was used to establish the antifungal efficacy of humic and fulvic acid extracted from vermi product against three Candida strains. Briefly, 100 µL of 3 h culture (approx. 1 × 10 6 CFU/mL) of each fungal strain was swabbed on YEPD agar plates using sterile cotton swab and allowed to air dry (aseptically) for 2 min. Then, four representative wells were made on YEPD agar plates using well puncher. The wells were then loaded with 25, 50, 75 and 100 µL of respective HA and FA samples and incubated for 24 h at the temperature of 37°C. Subsequent to incubation, the inhibitory propensity of extracted HA and FA was ascertained on the Candida species growth through the formation of inhibitory zone around the loaded wells. Results and discussion With practice valuable technology-based protocol, a mixture of sugarcane waste, coconut husk, leaf litter and vegetable waste with cow dung using Perionx excavatus was converted to an organically rich vermicompost. On 60th day of experiment, the granular, odourless and nonsticky vermicompost, granular vermicast and vermiwash were collected. The FA extraction rate is increased depending on the solvent ability to make complexes with metal cations boned with the group of phenolic and then carboxyl (De Paolis and Kukkonen, 1997 ; Huey, 2010 ). The dark brown or black colour may be caused by the presence of melanin and elemental configuration in HA and FA that was extracted from organic substances, vermicompost, vermicast and vermiwash. The impact of HA on the growth of the plant and its constituents has been examined in numerous research works. The outcome of FA foliar application on wheat yield, water utilization and nutrient uptake has also been described previously (Lu and Liu, 1986 ). Furthermore, in tomato plants, increases in leaf chlorophyll, diameter, stem height, size and production was observed and reported (Xudan, 1986 ; Yang et al., 2004 ). Humic acids extracted from vermicompost improved ATPase activity, plasma membrane H + , lateral root emergence and root elongation in maize (Böhme, 1999 ). Therefore, characterization and evaluation of HA and FA when used as fertilizer is considered crucial. The scientific data from this study may be utilized to determine the suitability of humic substances of HA and FA for usage as good fertilizer. All HA and FA samples are rich in micronutrients especially rich in copper (Cu) that are beneficial for the plant’s growth and survival. FT-IR analysis The important functional groups in the HA and FA were acknowledged by using the FTIR spectral tools. It’s also used to understand the structural changes in HA and FA (Egli et al., 2010 ; Tatzber et al., 2009 ). Significant differences in the major peaks in the spectra were not witnessed for HA and FA extracted from different samples. Based on the results, it was noted that the functional groups were common in all vermiproduct samples. Moderate and strong intensities of FTIR Spectra in OH stretch, aromatic CH out of plane bending, CH 3 OH of phenols, COO¯, ethers and esters, C-O of alcohols, phosphonates in each HA and FA samples are indicating the high potential to bind with metallic species (Laird et al., 2001 ; Skoog et al., 1985 ). In Fig. 1 Fig. 2 , the FTIR spectra plot of HA and FA are illustrated. The HA and FA isolated from vermicompost, vermicast, and vermiwash samples showed very similar FTIR spectra. In all of them, around 3300–3500 cm − 1 of broad band conforming the alcohol and phenol O-H stretching frequencies (Maccarthy and Rice, 1985 ; Mendonça et al., 2004 ). The appearance of three different weak bands at 2362, 2359, and 2341 cm − 1 suggested phosphonates. The asymmetric -COO stretch of carboxyl groups found at various places in the HA and FA is assigned in the range between 1550 and 1650 cm − 1 . A peak around 1290 − 1220 cm − 1 corresponds to C-O stretch and OH deformation, whereas the peak intensity increases at about 1421 cm − 1 corresponds to COO¯ stretching in carboxylic acids. A band between 1180 and 1000 cm − 1 is allocated to the (C-O) of alcohols, esters and ethers in the HA and FA samples. A beak between 900 and 650 cm − 1 represents aromatic CH out of plane bending. The appearance of the peaks between 779 and 460 cm − 1 with slight variations, all of the samples have a similar pattern of peaks that may be attributed to polysulfide groups and alkyl halides (Francioso et al., 1998 ; Hemati et al., 2012 ; Stevenson, 1995 ). SEM analysis Figures 3 and 4 shows the granular structure of humic and fulvic acid that have been extracted from three main sources of vermiproducts, i.e., vermicompost, vermicast, and vermiwash. Above all, they appear in crystal pattern accumulation besides many shapes. The SEM results are in good correlation with various crystals that have been discovered in XRD method for all HA and FA samples. The larger number of surface changes shown in SEM images of vermicompost samples indicate that the earthworms were involved in the biodegradation and mineralization of trash. The reduction of negative charges in HA structures, hydrogen bonds between humic substances that makes macromolecular configuration and weak electrostatic forces contributed to the aggregation of HA in acidic pH; as well as flexible of HA in alkaline pH is related to functional group ionization to generate strong electrostatic repulsion and breakdown of the weak electrostatic forces makes scattered configuration was shown (Pontié et al., 2012 ; Saab et al., 2010 ; Zara et al., 2006 ). The present study also showed the same results that observed in oven dried HA and FA samples. The plots of studied HA and FA showed precipitated and highly compacted structure with some extent of even forms and some of the images are heterogeneity due to particle distribution. E4/E6 ratio The E4/E6 ratio is an essential and very informative to characterize the aromaticity of humic substances. The HA extracted from vermicast showed relatively high E4/E6 ratio than vermicompost and vermiwash and it was in correspondence with the findings of Rajashekhar ( 2017 ). The UV-Vis spectra retrieved in this study for HA and FA were largely specific, with the absorbance decreasing as the wavelength increased (Fig. 5 ). All the absorption spectra showed similar pattern to HA and FA. The absorbances of peak between 250 and 400 nm have attributed significantly the nature of the HA. The E4/E6 ratio is a reliable and useful study for describing the aromaticity of HA and FA compounds (Kononova, 1966 ). In the present study, HA extracted from vermicasts had the highest E4/E6 ratio (6.1), followed by vermiwash (5.01). The E4/E6 ratio was slightly lower in vermicompost (4.95). In FA the highest E4/E6 ratio was observed in vermicompost (6.08) followed by vermiwash (5.59), and the lowest E4/E6 ratio was recorded in vermicast. (4.56) (Fig. 6 ) the highest value of E4/E6 refers to more aliphatic nature of the sample fractions (Garcia et al., 1991 ). At the same time, it reflects a lowest aromatic condensation. Similar results were reported by Kadalli et al. ( 2000 ). The (UV-Vis) spectra of FA and HA from three organic substances (vermicompost, vermicast, vermiwash) increasing wavelengths while decreasing absorbance. In this study, HA and FA showed a small peak around 280 nm, which was evident in the 6 samples due to the aromatic systems such as aniline phenolic groups, polyenes and polycyclic aromatic hydrocarbons (Rodríguez et al., 2016 ). Most of all HA and FA samples showed absorbance’s at 380 nm. In this, previousy mentioned characteristic nature of humic materials and results for peaks overlapping corresponding to various group of chromophoric in addition to aromatic and conjugation ensuring the peaks splinting as well as the connection of the same in a misshapen spectrum (Maccarthy and Rice, 1985 ). XRD analysis The phase formation and crystalline nature of the HA and FA extracted from vermicompost, vermicast and vermiwash were examined by powder XRD in Xpert Pro X-ray diffractometer using Cu K α radiation ( λ = 1.54060 Å). The results as illustrated in Fig. 7 and Fig. 8 conclude that the materials were well crystallized with sharp diffraction peaks at 2θ values 32 θ, 45 θ which are assigned to corresponding planes. There were some slight differences in the sorts of crystals observed in all sample may be due to physicochemical factors (Arroyo et al., 2004 ; Yamauchi et al., 2004 ). Antifungal susceptibility assays To ascertain the anti-candidal efficacy of HA and FA extracts on planktonic growth of Candida species, agar well diffusion assay was performed. For this, three clinically important Candida species, including, C. glabrata , C. tropicalis and C. albicans were utilized as they account for more than 80% of candidiasis worldwide (Roscetto et al., 2018 ). After incubation of fungal strains with various concentrations of extracted samples i.e., 25, 50, 75 and 100 µL, the anti-candida efficacy of HA and FA extracted from vermiproducts was ascertained through the formation of zone of clearance. As shown in Fig. 9 , the zone of inhibition was observed in the wells that are loaded with HA extracted from vermicompost, vermicast, vermiwash (HAVC, HAVCa and HAVW). This demonstrated that the HA extract (HAVC, HAVCa and HAVW) have the remarkable potency to inhibit the growth of all three species of Candida , which in turn confirms the proficient anti-candidal activity of this extract. At the same time, the FA extracted from vermicompost, vermicast, and vermiwash (FAVC, FAVCa and FAVW) did not impose any considerable effect on the growth of Candida species, which suggests the non-fungicidal effect of FA extracted from vermiproduct against all the used strains. Table 1 shows the wide spectrum anti-candidal activity of HA extracts from vermicompost, vermicast, and vermiwash (HAVC, HAVCa and HAVW). It was also observed that increase in volume of the extracts portrayed increase in the zone of clearance, which obviously signified that the inhibitory propensity of HA extract was in the concentration dependent fashion. Table 1 The zone of inhibition (mm) of six extracts against three clinically important Candida species. ± indicates the standard deviation of three independent experimental duplicates. N signifies “no zone of inhibition”. Test strains Volume of extracts Zone of inhibition (mm) Humic acid extract Fulvic acid extract HAVC HAVCa HAVW FAVC FAVCa FAVW C. albicans 25 µL 3 ± 0.2 12 ± 1.5 13 ± 1.2 N N N 50 µL 3 ± 0.3 14 ± 1.4 15 ± 1.3 75 µL 18 ± 2.0 19 ± 1.3 20 ± 0.9 100 µL 19 ± 1.4 21 ± 0.5 21 ± 0.7 C. tropicalis 25 µL N N 2 ± 0.5 N N N 50 µL N N 11 ± 0.8 75 µL 2 ± 0.3 2 ± 0.1 13 ± 1.2 100 µL 12 ± 1.3 13 ± 1.4 15 ± 1.1 C. glabrata 25 µL 9 ± 1.4 8 ± 0.6 5 ± 0.6 N N N 50 µL 10 ± 1.0 9 ± 0.8 12 ± 0.9 75 µL 12 ± 1.2 19 ± 1.4 21 ± 1.5 100 µL 15 ± 1.5 22 ± 0.9 23 ± 1.8 N signifies “no zone of inhibition” Other investigators have previously reported the prevalence rates of C. tropicalis , C. albicans , and C. glabrata in oral infections, and they have also reported that the Candida species linked to chronic periodontitis are susceptible to fluconazole. Similar results were obtained from this study also. The analytical results from this detailed study demonstrate that fulvic and humic acids were well characterized by various techniques. The analysis done in this study is helpful in highlighting slight variations in fulvic and humic acids isolated from the different organic sources, such as, vermicompost, vermicast, and vermiwash. Conclusions The optimal yield of fulvic and humic acids can be attained with an extraction time of 24 h. Additionally, 4 h are needed to precipitate the HA and FA samples in 1 h with distilled water without changing their real nature. The absorbance in the UV spectra bands was visible at 270 to 390 nm. The HA extracted from vermicast showed relatively high E4/E6 ratio, whereas in FA, the highest E4/E6 ratio was observed in vermicompost. FTIR bands ranging from 3300–3500 cm − 1 represent O-H stretching frequencies of alcohol and phenol, 1550–1650 cm − 1 represent asymmetric COO¯ stretch of carboxyl groups, 1290 − 1220 cm − 1 represent C-O stretch and O-H deformation, represent 1421 cm − 1 COO¯ stretching – carboxylic acids, 1180 − 1000 cm − 1 represent C-O of alcohols, esters and ethers, 900 − 650 cm − 1 represent plane bending of aromatic CH, 779 and 460 cm − 1 represent alkyl halides and polysulfide groups were clearly observed. The results are in agreement with the crystals that have been found previously using X-Ray diffraction methods. The SEM images show the granular structure of fulvic and humic acids. Therefore, the results suggest that HA and FA extracted from vermiproduct can be developed as potential antifungal therapeutic agent of treatment against Candida associated skin infections. Declarations Acknowledgements The authors acknowledge Alagappa University, Karaikkudi for laboratory facilities and encouragement and the authors also acknowledge Rashtriya Ucchatar Shiksha Abhiyan (RUSA) –Phase 2.0 grant, New Delhi (F. 24-51/2014U). Author Contributions K.V. and K.K.: Material preparation, data collection and analysis, Drafting the article. J.A.J.P.: Methodology, Drafting the article, Data analysis and interpretation. P.K.: Formal analysis, Investigation, Data collection. S.G. : Study conception and design; Data analysis and interpretation; R.B ., D.P., and S.R.: Data analysis and interpretation, Critical revision of the article. M.B .: Supervision, Data analysis and interpretation, Critical revision of the article. All authors reviewed and approved the manuscript. Data Availability Statement All data generated or analysed during this study are included in this published article. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Adani, F., Genevini, P., Tambone, F., Montoneri, E., 2006. Compost effect on soil humic acid: A NMR study. Chemosphere 65, 1414–1418. https://doi.org/10.1016/j.chemosphere.2006.03.070 Arroyo, L.J., Li, H., Teppen, B.J., Johnston, C.T., Boyd, S.A., 2004. Hydrolysis of carbaryl by carbonate impurities in reference clay SWy-2. J. Agric. Food Chem. 52, 8066–8073. https://doi.org/10.1021/jf048971b Atiyeh, R., Lee, S., Edwards, C., Arancon, N., Metzger, J., 2002. The influence of humic acids derived from earthworm-processed organic wastes on plant growth. Bioresour. Technol. 84, 7–14. https://doi.org/10.1016/S0960-8524(02)00017-2 Bhat, S.A., Singh, J., Vig, A.P., 2014. Genotoxic assessment and optimization of pressmud with the help of exotic earthworm Eisenia fetida . Environ. Sci. Pollut. Res. 21, 8112–8123. https://doi.org/10.1007/s11356-014-2758-2 Böhme, M., 1999. Effects of lactate, humate and Bacillus subtilis on the growth of tomato plants in hydroponic systems. Acta Hortic. 231–240. https://doi.org/10.17660/ActaHortic.1999.481.24 Campitelli, P., Ceppi, S., 2008. Effects of composting technologies on the chemical and physicochemical properties of humic acids. Geoderma 144, 325–333. https://doi.org/10.1016/j.geoderma.2007.12.003 De Paolis, F., Kukkonen, J., 1997. Binding of organic pollutants to humic and fulvic acids: Influence of pH and the structure of humic material. Chemosphere 34, 1693–1704. https://doi.org/10.1016/S0045-6535(97)00026-X Dores-Silva, P.R., Landgraf, M.D., Rezende, M.O. de O., 2013. Processo de estabilização de resíduos orgânicos: vermicompostagem versus compostagem. Quim. Nova 36, 640–645. https://doi.org/10.1590/S0100-40422013000500005 Egli, M., Mavris, C., Mirabella, A., Giaccai, D., 2010. Soil organic matter formation along a chronosequence in the Morteratsch proglacial area (Upper Engadine, Switzerland). Catena 82, 61–69. https://doi.org/10.1016/j.catena.2010.05.001 Francioso, O., Sánchez-Cortés, S., Tugnoli, V., Ciavatta, C., Gessa, C., 1998. Characterization of peat fulvic acid fractions by means of FT-IR, SERS, and 1 H, 13 C NMR spectroscopy. Appl. Spectrosc. 52, 270–277. https://doi.org/10.1366/0003702981943347 Garcia, J., Hernandez, J., Costa, F., 1991. Characterization of humic fractions from a municipal solid waste during composting. SueloY. Planta 1, 43–48. Gonelimali, F.D., Lin, J., Miao, W., Xuan, J., Charles, F., Chen, M., Hatab, S.R., 2018. Antimicrobial properties and mechanism of action of some plant extracts against food pathogens and spoilage microorganisms. Front. Microbiol. 9. https://doi.org/10.3389/fmicb.2018.01639 Hanisah, M.R.N., Ahmed, O.H., Susilawati, K., Majid, N.M.A., Jalloh, M.B., 2008. Determination of minimal duration essential for isolation of humic acids from soils in forest restoration programmes. Am. J. Agric. Biol. Sci. 3, 597–601. https://doi.org/10.3844/ajabssp.2008.597.601 Hemati, A., Alikhani, H., Marandi, G., 2012. Extractants and extraction time effects on physicochemical properties of humic acid. Intl. J. Agric Res Rev 2, 975–984. Hoitink, H.A.J., Fahy, P.C., 1986. Basis for the control of soilborne plant pathogens with composts. Annu. Rev. Phytopathol. 24, 93–114. https://doi.org/10.1146/annurev.py.24.090186.000521 Huey, 2010. Effects of extractants on the yields and selected chemical characteristics of humic acids isolated from tropical saprists peat. Am. J. Appl. Sci. 7, 933–936. https://doi.org/10.3844/ajassp.2010.933.936 Inbar, Y., Chen, Y., Hadar, Y., 1990. Humic substances formed during the composting of organic matter. Soil Sci. Soc. Am. J. 54, 1316–1323. https://doi.org/10.2136/sssaj1990.03615995005400050019x Ismail, S., 2005. The earthworm book. Other India Press. Mapusa 101. Kadalli, G., Suseela, D., Siddaramappa, R., John, E., 2000. Characterization of humic fractions extracted from coirdust-based composts. J Indian Soc. Soil. Sci. 48, 51–55. Kalsom, M.S.., Nur, H., Norlea, A.A., Ngaspan, S., 2006. Characterization of humic acid from humification of oil palm empty fruit bunch fibre using Trichoderma viride . J. Trop. Agric. Food Sci. 34, 165–172. Kononova, M.M., 1966. Soil organic matter: Its nature, its role in soil formation and in soil fertility. Pergamon Press Ltd., Oxford 45–49. Laird, D.A., Martens, D.A., Kingery, W.L., 2001. Nature of clay‐humic complexes in an agricultural soil. Soil Sci. Soc. Am. J. 65, 1413–1418. https://doi.org/10.2136/sssaj2001.6551413x Lu, F.J., Liu, T.M., 1986. Fluorescent compounds in drinking water of blackfoot disease endemic areas: animal experimental model. Taiwan Yi Xue Hui Za Zhi. 85, 352–8. Maccarthy, P., Rice, J.A., 1985. Spectroscopic methods (other than NMR) for determining functionality in humic substances. Humic Subst. Soil, Sediment Water. 527–543. McQuilken, M.P., Whipps, J.M., Lynch, J.M., 1994. Effects of water extracts of a composted manure-straw mixture on the plant pathogen Botrytis cinerea . World J. Microbiol. Biotechnol. 10, 20–26. https://doi.org/10.1007/BF00357556 Mendonça, A., Duarte, A.C., Santos, E.B.H., 2004. Spectroscopic properties of sedimentary humic acids from a salt marsh (Ria de Aveiro, Portugal): comparison of sediments colonized by Halimione portulacoides (L.) Aellen and non-vegetated sediments. Biogeochemistry 69, 159–174. https://doi.org/10.1023/B:BIOG.0000031078.38692.62 Mignone, R.A., Martin, M. V., Morán Vieyra, F.E., Palazzi, V.I., López de Mishima, B., Mártire, D.O., Borsarelli, C.D., 2012. Modulation of optical properties of dissolved humic substances by their molecular complexity †. Photochem. Photobiol. 88, 792–800. https://doi.org/10.1111/j.1751-1097.2012.01135.x Myneni, S.C.B., Brown, J.T., Martinez, G.A., Meyer-Ilse, W., 1999. Imaging of humic substance macromolecular structures in water and soils. Science (80-. ). 286, 1335–1337. https://doi.org/10.1126/science.286.5443.1335 Nifant’eva, T.I., Shkinev, V.M., Ya. Spivakov, B., 1994. In: D.L. Pyle (Ed.), Separations for Biotechnology. SCI, Cambridge 358. Nogales, R., Cifuentes, C., Benítez, E., 2005. Vermicomposting of winery wastes: A laboratory study. J. Environ. Sci. Heal. Part B 40, 659–673. https://doi.org/10.1081/PFC-200061595 Pontié, M., Thekkedath, A., Kecili, K., Dach, H., De Nardi, F., Castaing, J.B., 2012. Clay filter-aid in ultrafiltration (UF) of humic acid solution. Desalination 292, 73–86. https://doi.org/10.1016/j.desal.2012.02.011 Rajashekhar, D., 2017. Functional and spectral characterization of humic fractions obtained from organic manures. Int. J. Pure Appl. Biosci. 5, 1254–1259. https://doi.org/10.18782/2320-7051.5454 Rodríguez, F.J., Schlenger, P., García-Valverde, M., 2016. Monitoring changes in the structure and properties of humic substances following ozonation using UV–Vis, FTIR and 1 H NMR techniques. Sci. Total Environ. 541, 623–637. https://doi.org/10.1016/j.scitotenv.2015.09.127 Roscetto, E., Contursi, P., Vollaro, A., Fusco, S., Notomista, E., Catania, M.R., 2018. Antifungal and anti-biofilm activity of the first cryptic antimicrobial peptide from an archaeal protein against Candida spp. clinical isolates. Sci. Rep. 8, 17570. https://doi.org/10.1038/s41598-018-35530-0 Saab, S. da C., Carvalho, E.R., Bernardes Filho, R., Moura, M.R. de, Martin-Neto, L., Mattoso, L.H.C., 2010. pH effect in aquatic fulvic acid from a Brazilian river. J. Braz. Chem. Soc. 21, 1490–1496. https://doi.org/10.1590/S0103-50532010000800012 Schnitzer, M., Preston, C.M., 1986. Analysis of humic acids by solution and solid‐state carbon‐13 nuclear magnetic resonance. Soil Sci. Soc. Am. J. 50, 326–331. https://doi.org/10.2136/sssaj1986.03615995005000020015x Skoog, D.A., Holler, F.J., Crouch, S.R., 1985. Principles of instrumental analysis. Saunders Coll. Publ. USA 344–347. Stevenson, F.J., 1995. Humus chemistry: Genesis, composition, reactions., Second Edi. ed, Journal of Chemical Education. https://doi.org/10.1021/ed072pA93.6 Tan, K.H., 2014. Humic matter in soil and the environment. CRC Press. https://doi.org/10.1201/b17037 Tatzber, M., Stemmer, M., Spiegel, H., Katzlberger, C., Zehetner, F., Haberhauer, G., Garcia-Garcia, E., Gerzabek, M.H., 2009. Spectroscopic behaviour of 14C-labeled humic acids in a long-term field experiment with three cropping systems. Soil Res. 47, 459. https://doi.org/10.1071/SR08231 Warman, P.R., AngLopez, M.J., 2010. Vermicompost derived from different feedstocks as a plant growth medium. Bioresour. Technol. 101, 4479–4483. https://doi.org/10.1016/j.biortech.2010.01.098 Watanabe, K., Manefield, M., Lee, M., Kouzuma, A., 2009. Electron shuttles in biotechnology. Curr. Opin. Biotechnol. 20, 633–641. https://doi.org/10.1016/j.copbio.2009.09.006 Xudan, X., 1986. The effect of foliar application of fulvic acid on water use, nutrient uptake and yield in wheat. Aust. J. Agric. Res. 37, 343. https://doi.org/10.1071/AR9860343 Yamauchi, N., Toyodome, W., Umeda, K., Nishida, N., Murae, T., 2004. Structural features of humic acid of the coastal sediment in Ariake sea tidelands: Use of humic acid as an environmental indicator for river basins and coastal regions. Anal. Sci. 20, 1453–1457. https://doi.org/10.2116/analsci.20.1453 Yang, C.-M., Wang, M.-C., Lu, Y.-F., Chang, I.-F., Chou, C.-H., 2004. Humic substances affect the activity of chlorophyllase. J. Chem. Ecol. 30, 1057–1065. https://doi.org/10.1023/B:JOEC.0000028467.82191.f9 Zara, L.F., Rosa, A.H., Toscano, I.A.S., Rocha, J.C., 2006. A structural conformation study of aquatic humic acid. J. Braz. Chem. Soc. 17, 1014–1019. https://doi.org/10.1590/S0103-50532006000500028 Zhao, H., Liu, Y., Cui, Z., Beattie, D., Gu, Y., Wang, Q., 2011. Design, synthesis, and biological activities of arylmethylamine substituted chlorotriazine and methylthiotriazine compounds. J. Agric. Food Chem. 59, 11711–11717. https://doi.org/10.1021/jf203383s Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4221685","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288252874,"identity":"46eb8c34-6e3e-4ef3-ab00-4d7a17825821","order_by":0,"name":"K. Vanimuthu","email":"","orcid":"","institution":"Alagappa University","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"","lastName":"Vanimuthu","suffix":""},{"id":288252875,"identity":"8f73c764-f025-4d60-93ac-828f0ad3a8b6","order_by":1,"name":"K. Kavitha","email":"","orcid":"","institution":"Alagappa University","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"","lastName":"Kavitha","suffix":""},{"id":288252876,"identity":"96fc7869-22c7-40f0-a640-82fe110a34c6","order_by":2,"name":"J. Arockia John Paul","email":"","orcid":"","institution":"Arumugam Pillai Seethai Ammal College","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Arockia John","lastName":"Paul","suffix":""},{"id":288252877,"identity":"151da25e-37f7-4563-994f-29b5b03b306a","order_by":3,"name":"P. Kumar","email":"","orcid":"","institution":"Alagappa University","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"","lastName":"Kumar","suffix":""},{"id":288252878,"identity":"1fe01d76-685c-4a09-b4b6-8e238b376084","order_by":4,"name":"S. Gowrishankar","email":"","orcid":"","institution":"Alagappa University","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"","lastName":"Gowrishankar","suffix":""},{"id":288252879,"identity":"d68b3de8-7c4b-416e-a62c-b927c5bde32b","order_by":5,"name":"R. Balachandar","email":"","orcid":"","institution":"Prathyusha Engineering College","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Balachandar","suffix":""},{"id":288252880,"identity":"34a79061-97c4-48d0-8a47-dbffb4484753","order_by":6,"name":"D. Prabhu","email":"","orcid":"","institution":"Karpagam Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"","lastName":"Prabhu","suffix":""},{"id":288252881,"identity":"dc5c9ee9-0209-4248-803d-9c8d9e5a9ff4","order_by":7,"name":"S. Rajamanikandan","email":"","orcid":"","institution":"Karpagam Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"","lastName":"Rajamanikandan","suffix":""},{"id":288252882,"identity":"7964adea-ae7b-4fc5-9588-769b8a3c94e2","order_by":8,"name":"M. Biruntha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACA2YwdYCBn73/+4cPQCYbO1FaEg4wSPYcMGOcAdLCTEgLA1SLwQwHM2YeEIeQFnN23mfShT/uyBtIMKQ9tvm1TZ6PmYHxw8cc3Fosm9nNpGckPDPcLt1w3Di377ZhGzMDs+TMbXgcdpiNTZon4TDjzjkHG6Rze24zArWwMfMSocV+w41kBmnLntv2RGtJ3HAjjU2a4cftRIJaLJvZmK150g4nz+w5w2zY23A7uY2ZsRmvX8z5jzHe5rE5bNvP3sP44Mef27bz25sPfviIRwsqYGwDkw3EqgeBP6QoHgWjYBSMgpECAJopTqzwTkzRAAAAAElFTkSuQmCC","orcid":"","institution":"Alagappa University","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"","lastName":"Biruntha","suffix":""}],"badges":[],"createdAt":"2024-04-05 08:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4221685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4221685/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54452881,"identity":"939f2216-f885-4e19-83a2-246c1522b528","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111523,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of humic acid from vermiproducts, (a) vermicompost, (b) vermicast and (c) vermiwash.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/74f8da65cfe9554dfb332feb.png"},{"id":54452879,"identity":"308ccb16-4e8b-4cb1-a69f-7c4f77cbd527","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131777,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of fulvic acid from vermiproducts, (a) vermicompost, (b) vermicast and (c) vermiwash.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/b33c30a3469566f75df0af38.png"},{"id":54452878,"identity":"4f4bcf08-9ab3-4eb2-84de-61c21f540ec0","added_by":"auto","created_at":"2024-04-10 18:29:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":849249,"visible":true,"origin":"","legend":"\u003cp\u003eSEM of humic acid fraction extracted from vermiproducts, (a) vermicompost, (b) vermicast and (c) vermiwash.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/1cc566b48573a31dcf363c5a.png"},{"id":54453599,"identity":"f069958a-16f8-4c1d-94ac-cbf5bc839e3d","added_by":"auto","created_at":"2024-04-10 18:37:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":693618,"visible":true,"origin":"","legend":"\u003cp\u003eSEM of fulvic acid fraction extracted from vermiproducts, (a) vermicompost, (b) vermicast, and (c) vermiwash.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/7fb4b758d5e3effe360a1987.png"},{"id":54452885,"identity":"d92aea68-61c6-4883-aa2e-4df53832ed1c","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245480,"visible":true,"origin":"","legend":"\u003cp\u003eUV Spectra of humic acid and fulvic acid fractions from vermiproducts.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/c60550afbece88ebb26851fd.png"},{"id":54452887,"identity":"bb2311bb-9648-42ae-8e05-209f5432930c","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":123192,"visible":true,"origin":"","legend":"\u003cp\u003eE4/E6 ratio of fulvic and humic acid extracted from vermiproducts.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/eeb71c81e886eab382c4527a.png"},{"id":54452882,"identity":"a0331068-cce9-43b7-a06a-a810307b20db","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":575117,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of humic acid from vermiproducts, (a) Vermicompost, (b) Vermicast and (c) Vermiwash.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/63cd02c691b3692f61dbbf37.png"},{"id":54452884,"identity":"ec027a58-5a5f-45fd-bb9b-f62c271ac03a","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":555781,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of fulvic acid from vermiproduct, (a) Vermicompost, (b) Vermicast and (c) Vermiwash.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/d7384ccac6a2e85aa5b3ab95.png"},{"id":54452883,"identity":"0b6ae233-d6eb-4cb9-94f6-5bad4b22d63f","added_by":"auto","created_at":"2024-04-10 18:29:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1631626,"visible":true,"origin":"","legend":"\u003cp\u003eThe representative plate image showcasing the effect of fulvic and humic acid extracts on the growth of three \u003cem\u003eCandida\u003c/em\u003e species such as \u003cem\u003eC. glabrata, C. tropicalis \u003c/em\u003eand \u003cem\u003eC. albicans, \u003c/em\u003edetermined through agar well diffusion technique. The wells marked with 1, 2, 3 and 4 contained 25, 50, 75 and 100 µL volume of extracts, respectively.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/936d893bbc944696575e00b4.png"},{"id":54454242,"identity":"cf792f58-7fb9-4fdf-b35a-b395436ff993","added_by":"auto","created_at":"2024-04-10 18:46:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3747487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4221685/v1/862e3cbb-ba23-45e7-9fd3-0cc5f671a65d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, Characterization and Antifungal Behavior of Humic Acid and Fulvic Acid Fractions from Biowaste Derived Vermiproducts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobial or chemical humiliation of plants and animal matter are the main sources to form humic acid substances (Myneni et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Humic acids are insoluble in acidic whereas soluble in high pH conditions in an alkaline solution and are dark brown or black in colour (Tan, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The process of forming humus is known as humification that forms through the organic matter decomposition. The presence of major acidic functional groups, specifically carboxylic and phenolic groups and /C, H/C and O/C atomic ratios are primarily determines the humus (Watanabe et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). From several resources, including coal, lignite, farmyard manure, vermicompost, etc., can be utilized for the extraction of humic acid. Hydrophilic groups in the humic acid encourage hydration and hence increase the soil\u0026rsquo;s capacity of retaining water. In soil and compost, it is the most efficient as well as the main component of humic substances. Vermicomposting involves employing earthworms with their flora that resides inside the digestive systems of them to convert new organic matter into stable organic matter (Atiyeh et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Campitelli and Ceppi, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dores-Silva et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nogales et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The humic acid (HA) and fulvic acid (FA) have physical and chemical characteristics extracted from various sites are dependent on large number of aspects smilarly the value of the original material. Humification process is a slow and progressive that can take thousands of years to complete in nature which leads to produce humic substances with various structural characteristics (Adani et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mignone et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The use of decomposed materials is one of the most popular methods in organic farming. The particular substances bring about extracts of humic liquid that are usually composed of proteins, humic materials, and amino acids also contain variety of nutrients such as micro and macro nutrients. Vermicompost made from chicken litter, bovine manure, sewage sludge, urban trash and other renewable and natural sources are considered as the main sources to obtain liquid humic extracts (Warman and AngLopez, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Humic acids provide essential nutrients to plants through the regulation of carbon cycle and release of nutrients from compressed soil (Kalsom et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Stevenson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe oxidation-related damage can be inhibited or limited by performing the alkaline solution extraction under nitrogen environment, but the extraction process is still too harsh. The yield of humic acids is considerably lower with mild extractants including, cation exchange resins and sodium pyrophosphate when compared with alkaline solutions at the same extraction period. The solubility also varies in different extractants may be caused by variations in the macromolecules chemical structures or by organic macromolecules produced physically that interact to mineral fractions via cation bridges (Stevenson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The accurate information regarding the varied properties of humic substances and their fractions provided by combining multiple extraction techniques and fractionation processes (Nifant\u0026rsquo;eva et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The maximum profit of HA extracted in Bintulu Sarawak from rehabitated forest, could be acquired in a period of 4 h of extraction were revealed (Hanisah et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the study, the discernible interaction between the extraction and the fractionation period was not reported. This is due to the fact that fractionation following acidification had no impact on the HA yield from the soils. However, the HA had to be precipitated from the restored forest soils for about 4 h is required for the purification using distilled water. Recent studies have shown that organic amendment can help prevent plant illnesses brought on by a variety of soil-borne phytopathogenic microbes, including fungus of different genera (Hoitink and Fahy, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In that some investigations explained humic acids are suppressing of pathogen but they are reasonable for practice as biofertilizers in liquid form furthermore have been used against plant pathogen (McQuilken et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The information regarding the fungicidal properties of humic acid, which is a component of vermiproducts like vermicompost, vermicast, and vermiwash is not available much in the literature.\u003c/p\u003e \u003cp\u003eTherefore, the aim of the work is to isolate, characterize and to find out the antifungal activities of HA and FA in the vermiproducts (vermicompost, vermicast and vermiwash produced from biowastes) using alkaline precipitation method recommended by IHSS (International Humic Substances Standard). The characterization and purification of humic and fulvic acid done by SEM, FTIR, XRD and UV is useful for the preparation of HA and FA solutions in the precise concentrations needed for the treatment of the human pathogenic \u003cem\u003eCandida\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVermicompost unit construction\u003c/h2\u003e \u003cp\u003eVermicomposting beds were constructed with the length of 10 m\u003csup\u003e3\u003c/sup\u003e, width of 8 and height of 3 m\u003csup\u003e3\u003c/sup\u003e in the shade place. The vermitech patterns were used to study the construction of vermicomposting units (Ismail, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Earthworm culturing concrete tanks were set up of 150\u0026times;100\u0026times;60 cm\u003csup\u003e3\u003c/sup\u003e. To provide efficient water drainage, the concrete modules contained drainage holes (2 \u0026times; 2 cm\u003csup\u003e2\u003c/sup\u003e) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of vermiproducts\u003c/h2\u003e \u003cp\u003eTo get worm-bed, four beds were used and roofs were placed over each bed to shield the vermicomposting beds from the sun light and rain. The 1 week aged cow dung and mixed biowastes (sugarcane waste, leaf litter, coconut husk) in 1:1 (v/v) was given as substrates for the earthworms (\u003cem\u003ePerionyx excavatus\u003c/em\u003e) in each vermibed. The vermicompost bed was set to 80% vermiwash drainage and collection system for collecting vermiwash (Stevenson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of humic and fulvic acid\u003c/h2\u003e \u003cp\u003eAbout 10 g of air-dried vermiproduct samples, including, vermicompost, vermicast, vermiwash was deliberated in to 250 ml conical flask with 50 ml of 0.1 N sodium hydroxide and kept undisturbed for 24 h. After centrifugation, supernatant solution with dark colour was collected and the extraction process of humic acid was done repeatedly using 50 ml of extractant each time. Supernatants were combined in a conical flask, and the coloured solution was kept for 15 minutes in a centrifuge at 15,000 rpm to remove all the fine debris. In a 250 ml beaker, the coloured supernatant was collected after the clay was discarded. The pH electrode was immersed in the collected dark coloured supernatant solution and slowly 2 \u003cem\u003eN\u003c/em\u003e HCl was added until it reaches pH of 2 to precipitate the humic acids. After giving it a thorough stir, it was let to retain for 24 h at the room temperature. Coagulate (HA), and supernatant (FA) were separately collected and on a hot air oven, the collected coagulate was dried.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSpectroscopic characterization of humic and fulvic acids\u003c/h2\u003e \u003cp\u003eAcidic functional groups, including, phenolic (-OH) and carboxylic (-COOH) groups of HA were determined (Inbar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In a Thermo Nicolet 380 Fourier transfer IR spectrometer, Fourier Transform Infrared (FTIR) spectra was recorded from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in KBr pellets which contained humic acids (0.50 mg) along with dry KBr of 200 mg (Schnitzer and Preston, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). FTIR spectra are regarded as HA and FA fingerprints for vermicompost, vermicast, and vermiwash. FTIR spectra results are evident that HA and FA isolated from vermiproducts contain distinctive functional groups and bonds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSEM analysis of humic and fulvic acid\u003c/h2\u003e \u003cp\u003eExtracted, dried HA and FA were maintained at 40 \u0026ordm;C and sent to USIC at Alagappa University for SEM observation. Instead of using acid, the samples of vermiproducts like vermicompost, vermicast, vermiwash (0.2 g) were produced for qualitative and quantitative analysis and examined using a SEM (Quanta FEG250-High Resolution) for their microstructure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eE4/E6 ratio\u003c/h2\u003e \u003cp\u003eHA and FA were evaluated for their level of humification and aromaticity using E4/E6 ratio. At 10 ml of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e M NaHCO\u003csub\u003e3\u003c/sub\u003e solution, a well-known quantity of the samples were taken and dissolved. The ratio and absorbance were recorded using UV-Vis NIR spectrophotometer at wavelength of 190 and 1100 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eX-ray diffraction method\u003c/h2\u003e \u003cp\u003eX PERT PRO powder X-Ray Diffractrometer (XRD) system was used for the analysis of XRD patterns in the vermiproduct samples (Bhat et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Crystalline structures present in the samples were analysed with software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFungicidal activity against human pathogen\u003c/h2\u003e \u003cp\u003eUsing mycelium growth assays, the \u003cem\u003ein vitro\u003c/em\u003e fungicidal activity of HA and FA against \u003cem\u003eCandida\u003c/em\u003e species were evaluated (Zhao et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After dissolved in DMSO, HA and FA were diluted to 50 mg/L concentration in water that contained an emulsifier with 200 ug/mL. After that, 1 mL of test solution was put on sterile Petri plates with a diameter of 9 cm and finally 9 mL of agar culture media was added. The controls were maintained in 9 ml of agar culture media with samples lacking HA and FA. The inocula with a diameter of 4 mm was taken from the borders of energetically growing colonies and placed in the middle of the plates. Finally, zone of inhibition was measured by diameter after 72 h of incubation at 25\u0026deg;C. The plates were maintained in triplicates and the efficiency of inhibition of the HA and FA was calculated in terms of their inhibition percent (%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStrains and culture conditions\u003c/h2\u003e \u003cp\u003eIn this present study, antifungal efficacy of HA and FA extracted from vermiproducts (vermicompost, vermicast, and vermiwash) was evaluated against three clinically important \u003cem\u003eCandida\u003c/em\u003e species \u003cem\u003eviz., Candida albicans\u003c/em\u003e (ATCC 10231; purchased from HiMedia, India), \u003cem\u003eCandida glabrata\u003c/em\u003e (MTCC 3019; procured from CSIR-IMTECH, Chandigarh, India) \u003cem\u003eand Candida tropicalis\u003c/em\u003e (MTCC 184; obtained from CSIR-IMTECH, Chandigarh, India). Strains were maintained in YEPD agar plate, from which, a loop-full of colony was used to inoculate YEPD broth at the temperature of 37\u0026deg;C for overnight in rotary shaker with 200 rpm agitation. To perform the agar well diffusion assay, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL (0.1 OD) cells were used to swab on YEPD agar plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal susceptibility assays\u003c/h2\u003e \u003cp\u003eAgar well diffusion technique previously designated by (Gonelimali et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with required modifications was used to establish the antifungal efficacy of humic and fulvic acid extracted from vermi product against three \u003cem\u003eCandida\u003c/em\u003e strains. Briefly, 100 \u0026micro;L of 3 h culture (approx. 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) of each fungal strain was swabbed on YEPD agar plates using sterile cotton swab and allowed to air dry (aseptically) for 2 min. Then, four representative wells were made on YEPD agar plates using well puncher. The wells were then loaded with 25, 50, 75 and 100 \u0026micro;L of respective HA and FA samples and incubated for 24 h at the temperature of 37\u0026deg;C. Subsequent to incubation, the inhibitory propensity of extracted HA and FA was ascertained on the \u003cem\u003eCandida\u003c/em\u003e species growth through the formation of inhibitory zone around the loaded wells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eWith practice valuable technology-based protocol, a mixture of sugarcane waste, coconut husk, leaf litter and vegetable waste with cow dung using \u003cem\u003ePerionx excavatus\u003c/em\u003e was converted to an organically rich vermicompost. On 60th day of experiment, the granular, odourless and nonsticky vermicompost, granular vermicast and vermiwash were collected. The FA extraction rate is increased depending on the solvent ability to make complexes with metal cations boned with the group of phenolic and then carboxyl (De Paolis and Kukkonen, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Huey, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The dark brown or black colour may be caused by the presence of melanin and elemental configuration in HA and FA that was extracted from organic substances, vermicompost, vermicast and vermiwash.\u003c/p\u003e \u003cp\u003eThe impact of HA on the growth of the plant and its constituents has been examined in numerous research works. The outcome of FA foliar application on wheat yield, water utilization and nutrient uptake has also been described previously (Lu and Liu, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Furthermore, in tomato plants, increases in leaf chlorophyll, diameter, stem height, size and production was observed and reported (Xudan, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHumic acids extracted from vermicompost improved ATPase activity, plasma membrane H\u003csup\u003e+\u003c/sup\u003e, lateral root emergence and root elongation in maize (B\u0026ouml;hme, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, characterization and evaluation of HA and FA when used as fertilizer is considered crucial. The scientific data from this study may be utilized to determine the suitability of humic substances of HA and FA for usage as good fertilizer. All HA and FA samples are rich in micronutrients especially rich in copper (Cu) that are beneficial for the plant\u0026rsquo;s growth and survival.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFT-IR analysis\u003c/h2\u003e \u003cp\u003eThe important functional groups in the HA and FA were acknowledged by using the FTIR spectral tools. It\u0026rsquo;s also used to understand the structural changes in HA and FA (Egli et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tatzber et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Significant differences in the major peaks in the spectra were not witnessed for HA and FA extracted from different samples. Based on the results, it was noted that the functional groups were common in all vermiproduct samples. Moderate and strong intensities of FTIR Spectra in OH stretch, aromatic CH out of plane bending, CH\u003csub\u003e3\u003c/sub\u003e OH of phenols, COO\u0026macr;, ethers and esters, C-O of alcohols, phosphonates in each HA and FA samples are indicating the high potential to bind with metallic species (Laird et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Skoog et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the FTIR spectra plot of HA and FA are illustrated. The HA and FA isolated from vermicompost, vermicast, and vermiwash samples showed very similar FTIR spectra. In all of them, around 3300\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of broad band conforming the alcohol and phenol O-H stretching frequencies (Maccarthy and Rice, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Mendon\u0026ccedil;a et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The appearance of three different weak bands at 2362, 2359, and 2341 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e suggested phosphonates. The asymmetric -COO stretch of carboxyl groups found at various places in the HA and FA is assigned in the range between 1550 and 1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A peak around 1290\u0026thinsp;\u0026minus;\u0026thinsp;1220 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to C-O stretch and OH deformation, whereas the peak intensity increases at about 1421 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to COO\u0026macr; stretching in carboxylic acids. A band between 1180 and 1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is allocated to the (C-O) of alcohols, esters and ethers in the HA and FA samples. A beak between 900 and 650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents aromatic CH out of plane bending. The appearance of the peaks between 779 and 460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with slight variations, all of the samples have a similar pattern of peaks that may be attributed to polysulfide groups and alkyl halides (Francioso et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hemati et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Stevenson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSEM analysis\u003c/h2\u003e \u003cp\u003eFigures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the granular structure of humic and fulvic acid that have been extracted from three main sources of vermiproducts, i.e., vermicompost, vermicast, and vermiwash. Above all, they appear in crystal pattern accumulation besides many shapes. The SEM results are in good correlation with various crystals that have been discovered in XRD method for all HA and FA samples. The larger number of surface changes shown in SEM images of vermicompost samples indicate that the earthworms were involved in the biodegradation and mineralization of trash. The reduction of negative charges in HA structures, hydrogen bonds between humic substances that makes macromolecular configuration and weak electrostatic forces contributed to the aggregation of HA in acidic pH; as well as flexible of HA in alkaline pH is related to functional group ionization to generate strong electrostatic repulsion and breakdown of the weak electrostatic forces makes scattered configuration was shown (Ponti\u0026eacute; et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Saab et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zara et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The present study also showed the same results that observed in oven dried HA and FA samples. The plots of studied HA and FA showed precipitated and highly compacted structure with some extent of even forms and some of the images are heterogeneity due to particle distribution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eE4/E6 ratio\u003c/h2\u003e \u003cp\u003eThe E4/E6 ratio is an essential and very informative to characterize the aromaticity of humic substances. The HA extracted from vermicast showed relatively high E4/E6 ratio than vermicompost and vermiwash and it was in correspondence with the findings of Rajashekhar (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The UV-Vis spectra retrieved in this study for HA and FA were largely specific, with the absorbance decreasing as the wavelength increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All the absorption spectra showed similar pattern to HA and FA. The absorbances of peak between 250 and 400 nm have attributed significantly the nature of the HA. The E4/E6 ratio is a reliable and useful study for describing the aromaticity of HA and FA compounds (Kononova, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). In the present study, HA extracted from vermicasts had the highest E4/E6 ratio (6.1), followed by vermiwash (5.01). The E4/E6 ratio was slightly lower in vermicompost (4.95). In FA the highest E4/E6 ratio was observed in vermicompost (6.08) followed by vermiwash (5.59), and the lowest E4/E6 ratio was recorded in vermicast. (4.56) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) the highest value of E4/E6 refers to more aliphatic nature of the sample fractions (Garcia et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). At the same time, it reflects a lowest aromatic condensation. Similar results were reported by Kadalli et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe (UV-Vis) spectra of FA and HA from three organic substances (vermicompost, vermicast, vermiwash) increasing wavelengths while decreasing absorbance. In this study, HA and FA showed a small peak around 280 nm, which was evident in the 6 samples due to the aromatic systems such as aniline phenolic groups, polyenes and polycyclic aromatic hydrocarbons (Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Most of all HA and FA samples showed absorbance\u0026rsquo;s at 380 nm. In this, previousy mentioned characteristic nature of humic materials and results for peaks overlapping corresponding to various group of chromophoric in addition to aromatic and conjugation ensuring the peaks splinting as well as the connection of the same in a misshapen spectrum (Maccarthy and Rice, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eXRD analysis\u003c/h2\u003e \u003cp\u003eThe phase formation and crystalline nature of the HA and FA extracted from vermicompost, vermicast and vermiwash were examined by powder XRD in Xpert Pro X-ray diffractometer using Cu K\u003csub\u003eα\u003c/sub\u003e radiation (\u003cem\u003eλ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.54060 \u0026Aring;). The results as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e conclude that the materials were well crystallized with sharp diffraction peaks at 2θ values 32 θ, 45 θ which are assigned to corresponding planes. There were some slight differences in the sorts of crystals observed in all sample may be due to physicochemical factors (Arroyo et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yamauchi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal susceptibility assays\u003c/h2\u003e \u003cp\u003eTo ascertain the anti-candidal efficacy of HA and FA extracts on planktonic growth of \u003cem\u003eCandida\u003c/em\u003e species, agar well diffusion assay was performed. For this, three clinically important \u003cem\u003eCandida\u003c/em\u003e species, including, \u003cem\u003eC. glabrata\u003c/em\u003e, \u003cem\u003eC. tropicalis\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e were utilized as they account for more than 80% of candidiasis worldwide (Roscetto et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After incubation of fungal strains with various concentrations of extracted samples i.e., 25, 50, 75 and 100 \u0026micro;L, the anti-candida efficacy of HA and FA extracted from vermiproducts was ascertained through the formation of zone of clearance. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the zone of inhibition was observed in the wells that are loaded with HA extracted from vermicompost, vermicast, vermiwash (HAVC, HAVCa and HAVW). This demonstrated that the HA extract (HAVC, HAVCa and HAVW) have the remarkable potency to inhibit the growth of all three species of \u003cem\u003eCandida\u003c/em\u003e, which in turn confirms the proficient anti-candidal activity of this extract. At the same time, the FA extracted from vermicompost, vermicast, and vermiwash (FAVC, FAVCa and FAVW) did not impose any considerable effect on the growth of \u003cem\u003eCandida\u003c/em\u003e species, which suggests the non-fungicidal effect of FA extracted from vermiproduct against all the used strains. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the wide spectrum anti-candidal activity of HA extracts from vermicompost, vermicast, and vermiwash (HAVC, HAVCa and HAVW). It was also observed that increase in volume of the extracts portrayed increase in the zone of clearance, which obviously signified that the inhibitory propensity of HA extract was in the concentration dependent fashion.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe zone of inhibition (mm) of six extracts against three clinically important \u003cem\u003eCandida\u003c/em\u003e species. \u0026plusmn; indicates the standard deviation of three independent experimental duplicates. N signifies \u0026ldquo;no zone of inhibition\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTest strains\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eVolume of extracts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eZone of inhibition (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eHumic acid extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eFulvic acid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHAVC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHAVCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHAVW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAVC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFAVCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFAVW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eC. glabrata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eN signifies \u0026ldquo;no zone of inhibition\u0026rdquo;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOther investigators have previously reported the prevalence rates of \u003cem\u003eC. tropicalis\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e, and \u003cem\u003eC. glabrata\u003c/em\u003e in oral infections, and they have also reported that the \u003cem\u003eCandida\u003c/em\u003e species linked to chronic periodontitis are susceptible to fluconazole. Similar results were obtained from this study also. The analytical results from this detailed study demonstrate that fulvic and humic acids were well characterized by various techniques. The analysis done in this study is helpful in highlighting slight variations in fulvic and humic acids isolated from the different organic sources, such as, vermicompost, vermicast, and vermiwash.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe optimal yield of fulvic and humic acids can be attained with an extraction time of 24 h. Additionally, 4 h are needed to precipitate the HA and FA samples in 1 h with distilled water without changing their real nature. The absorbance in the UV spectra bands was visible at 270 to 390 nm. The HA extracted from vermicast showed relatively high E4/E6 ratio, whereas in FA, the highest E4/E6 ratio was observed in vermicompost. FTIR bands ranging from 3300\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent O-H stretching frequencies of alcohol and phenol, 1550\u0026ndash;1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent asymmetric COO\u0026macr; stretch of carboxyl groups, 1290\u0026thinsp;\u0026minus;\u0026thinsp;1220 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent C-O stretch and O-H deformation, represent 1421 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e COO\u0026macr; stretching \u0026ndash; carboxylic acids, 1180\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent C-O of alcohols, esters and ethers, 900\u0026thinsp;\u0026minus;\u0026thinsp;650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent plane bending of aromatic CH, 779 and 460 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent alkyl halides and polysulfide groups were clearly observed. The results are in agreement with the crystals that have been found previously using X-Ray diffraction methods. The SEM images show the granular structure of fulvic and humic acids. Therefore, the results suggest that HA and FA extracted from vermiproduct can be developed as potential antifungal therapeutic agent of treatment against \u003cem\u003eCandida\u003c/em\u003e associated skin infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Alagappa University, Karaikkudi for laboratory facilities and encouragement and the authors also acknowledge Rashtriya Ucchatar Shiksha Abhiyan (RUSA) \u0026ndash;Phase 2.0 grant, New Delhi (F. 24-51/2014U).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK.V. and K.K.:\u0026nbsp;\u003c/strong\u003eMaterial preparation, data collection and analysis, Drafting the article. \u003cstrong\u003eJ.A.J.P.:\u0026nbsp;\u003c/strong\u003eMethodology, Drafting the article, Data analysis and interpretation. \u003cstrong\u003eP.K.:\u0026nbsp;\u003c/strong\u003eFormal analysis, Investigation, Data collection. \u003cstrong\u003eS.G.\u003c/strong\u003e: Study conception and design; Data analysis and interpretation; \u003cstrong\u003eR.B\u003c/strong\u003e\u003cstrong\u003e., D.P., and S.R.:\u003c/strong\u003e Data analysis and interpretation, Critical revision of the article. \u003cstrong\u003eM.B\u003c/strong\u003e.: Supervision, Data analysis and interpretation, Critical revision of the article. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdani, F., Genevini, P., Tambone, F., Montoneri, E., 2006. Compost effect on soil humic acid: A NMR study. Chemosphere 65, 1414\u0026ndash;1418. https://doi.org/10.1016/j.chemosphere.2006.03.070\u003c/li\u003e\n\u003cli\u003eArroyo, L.J., Li, H., Teppen, B.J., Johnston, C.T., Boyd, S.A., 2004. Hydrolysis of carbaryl by carbonate impurities in reference clay SWy-2. J. Agric. Food Chem. 52, 8066\u0026ndash;8073. https://doi.org/10.1021/jf048971b\u003c/li\u003e\n\u003cli\u003eAtiyeh, R., Lee, S., Edwards, C., Arancon, N., Metzger, J., 2002. The influence of humic acids derived from earthworm-processed organic wastes on plant growth. Bioresour. Technol. 84, 7\u0026ndash;14. https://doi.org/10.1016/S0960-8524(02)00017-2\u003c/li\u003e\n\u003cli\u003eBhat, S.A., Singh, J., Vig, A.P., 2014. Genotoxic assessment and optimization of pressmud with the help of exotic earthworm \u003cem\u003eEisenia fetida\u003c/em\u003e. Environ. Sci. Pollut. Res. 21, 8112\u0026ndash;8123. https://doi.org/10.1007/s11356-014-2758-2\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;hme, M., 1999. Effects of lactate, humate and \u003cem\u003eBacillus subtilis\u003c/em\u003e on the growth of tomato plants in hydroponic systems. Acta Hortic. 231\u0026ndash;240. https://doi.org/10.17660/ActaHortic.1999.481.24\u003c/li\u003e\n\u003cli\u003eCampitelli, P., Ceppi, S., 2008. Effects of composting technologies on the chemical and physicochemical properties of humic acids. Geoderma 144, 325\u0026ndash;333. https://doi.org/10.1016/j.geoderma.2007.12.003\u003c/li\u003e\n\u003cli\u003eDe Paolis, F., Kukkonen, J., 1997. Binding of organic pollutants to humic and fulvic acids: Influence of pH and the structure of humic material. Chemosphere 34, 1693\u0026ndash;1704. https://doi.org/10.1016/S0045-6535(97)00026-X\u003c/li\u003e\n\u003cli\u003eDores-Silva, P.R., Landgraf, M.D., Rezende, M.O. de O., 2013. Processo de estabiliza\u0026ccedil;\u0026atilde;o de res\u0026iacute;duos org\u0026acirc;nicos: vermicompostagem versus compostagem. Quim. Nova 36, 640\u0026ndash;645. https://doi.org/10.1590/S0100-40422013000500005\u003c/li\u003e\n\u003cli\u003eEgli, M., Mavris, C., Mirabella, A., Giaccai, D., 2010. Soil organic matter formation along a chronosequence in the Morteratsch proglacial area (Upper Engadine, Switzerland). Catena 82, 61\u0026ndash;69. https://doi.org/10.1016/j.catena.2010.05.001\u003c/li\u003e\n\u003cli\u003eFrancioso, O., S\u0026aacute;nchez-Cort\u0026eacute;s, S., Tugnoli, V., Ciavatta, C., Gessa, C., 1998. Characterization of peat fulvic acid fractions by means of FT-IR, SERS, and 1 H, 13 C NMR spectroscopy. Appl. Spectrosc. 52, 270\u0026ndash;277. https://doi.org/10.1366/0003702981943347\u003c/li\u003e\n\u003cli\u003eGarcia, J., Hernandez, J., Costa, F., 1991. Characterization of humic fractions from a municipal solid waste during composting. SueloY. Planta 1, 43\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eGonelimali, F.D., Lin, J., Miao, W., Xuan, J., Charles, F., Chen, M., Hatab, S.R., 2018. Antimicrobial properties and mechanism of action of some plant extracts against food pathogens and spoilage microorganisms. Front. Microbiol. 9. https://doi.org/10.3389/fmicb.2018.01639\u003c/li\u003e\n\u003cli\u003eHanisah, M.R.N., Ahmed, O.H., Susilawati, K., Majid, N.M.A., Jalloh, M.B., 2008. Determination of minimal duration essential for isolation of humic acids from soils in forest restoration programmes. Am. J. Agric. Biol. Sci. 3, 597\u0026ndash;601. https://doi.org/10.3844/ajabssp.2008.597.601\u003c/li\u003e\n\u003cli\u003eHemati, A., Alikhani, H., Marandi, G., 2012. Extractants and extraction time effects on physicochemical properties of humic acid. Intl. J. Agric Res Rev 2, 975\u0026ndash;984.\u003c/li\u003e\n\u003cli\u003eHoitink, H.A.J., Fahy, P.C., 1986. Basis for the control of soilborne plant pathogens with composts. Annu. Rev. Phytopathol. 24, 93\u0026ndash;114. https://doi.org/10.1146/annurev.py.24.090186.000521\u003c/li\u003e\n\u003cli\u003eHuey, 2010. Effects of extractants on the yields and selected chemical characteristics of humic acids isolated from tropical saprists peat. Am. J. Appl. Sci. 7, 933\u0026ndash;936. https://doi.org/10.3844/ajassp.2010.933.936\u003c/li\u003e\n\u003cli\u003eInbar, Y., Chen, Y., Hadar, Y., 1990. Humic substances formed during the composting of organic matter. Soil Sci. Soc. Am. J. 54, 1316\u0026ndash;1323. https://doi.org/10.2136/sssaj1990.03615995005400050019x\u003c/li\u003e\n\u003cli\u003eIsmail, S., 2005. The earthworm book. Other India Press. Mapusa 101.\u003c/li\u003e\n\u003cli\u003eKadalli, G., Suseela, D., Siddaramappa, R., John, E., 2000. Characterization of humic fractions extracted from coirdust-based composts. J Indian Soc. Soil. Sci. 48, 51\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eKalsom, M.S.., Nur, H., Norlea, A.A., Ngaspan, S., 2006. Characterization of humic acid from humification of oil palm empty fruit bunch fibre using \u003cem\u003eTrichoderma viride\u003c/em\u003e. J. Trop. Agric. Food Sci. 34, 165\u0026ndash;172.\u003c/li\u003e\n\u003cli\u003eKononova, M.M., 1966. Soil organic matter: Its nature, its role in soil formation and in soil fertility. Pergamon Press Ltd., Oxford 45\u0026ndash;49.\u003c/li\u003e\n\u003cli\u003eLaird, D.A., Martens, D.A., Kingery, W.L., 2001. Nature of clay‐humic complexes in an agricultural soil. Soil Sci. Soc. Am. J. 65, 1413\u0026ndash;1418. https://doi.org/10.2136/sssaj2001.6551413x\u003c/li\u003e\n\u003cli\u003eLu, F.J., Liu, T.M., 1986. Fluorescent compounds in drinking water of blackfoot disease endemic areas: animal experimental model. Taiwan Yi Xue Hui Za Zhi. 85, 352\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eMaccarthy, P., Rice, J.A., 1985. Spectroscopic methods (other than NMR) for determining functionality in humic substances. Humic Subst. Soil, Sediment Water. 527\u0026ndash;543.\u003c/li\u003e\n\u003cli\u003eMcQuilken, M.P., Whipps, J.M., Lynch, J.M., 1994. Effects of water extracts of a composted manure-straw mixture on the plant pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e. World J. Microbiol. Biotechnol. 10, 20\u0026ndash;26. https://doi.org/10.1007/BF00357556\u003c/li\u003e\n\u003cli\u003eMendon\u0026ccedil;a, A., Duarte, A.C., Santos, E.B.H., 2004. Spectroscopic properties of sedimentary humic acids from a salt marsh (Ria de Aveiro, Portugal): comparison of sediments colonized by \u003cem\u003eHalimione portulacoides\u003c/em\u003e (L.) Aellen and non-vegetated sediments. Biogeochemistry 69, 159\u0026ndash;174. https://doi.org/10.1023/B:BIOG.0000031078.38692.62\u003c/li\u003e\n\u003cli\u003eMignone, R.A., Martin, M. V., Mor\u0026aacute;n Vieyra, F.E., Palazzi, V.I., L\u0026oacute;pez de Mishima, B., M\u0026aacute;rtire, D.O., Borsarelli, C.D., 2012. Modulation of optical properties of dissolved humic substances by their molecular complexity \u0026dagger;. Photochem. Photobiol. 88, 792\u0026ndash;800. https://doi.org/10.1111/j.1751-1097.2012.01135.x\u003c/li\u003e\n\u003cli\u003eMyneni, S.C.B., Brown, J.T., Martinez, G.A., Meyer-Ilse, W., 1999. Imaging of humic substance macromolecular structures in water and soils. Science (80-. ). 286, 1335\u0026ndash;1337. https://doi.org/10.1126/science.286.5443.1335\u003c/li\u003e\n\u003cli\u003eNifant\u0026rsquo;eva, T.I., Shkinev, V.M., Ya. Spivakov, B., 1994. In: D.L. Pyle (Ed.), Separations for Biotechnology. SCI, Cambridge 358.\u003c/li\u003e\n\u003cli\u003eNogales, R., Cifuentes, C., Ben\u0026iacute;tez, E., 2005. Vermicomposting of winery wastes: A laboratory study. J. Environ. Sci. Heal. Part B 40, 659\u0026ndash;673. https://doi.org/10.1081/PFC-200061595\u003c/li\u003e\n\u003cli\u003ePonti\u0026eacute;, M., Thekkedath, A., Kecili, K., Dach, H., De Nardi, F., Castaing, J.B., 2012. Clay filter-aid in ultrafiltration (UF) of humic acid solution. Desalination 292, 73\u0026ndash;86. https://doi.org/10.1016/j.desal.2012.02.011\u003c/li\u003e\n\u003cli\u003eRajashekhar, D., 2017. Functional and spectral characterization of humic fractions obtained from organic manures. Int. J. Pure Appl. Biosci. 5, 1254\u0026ndash;1259. https://doi.org/10.18782/2320-7051.5454\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez, F.J., Schlenger, P., Garc\u0026iacute;a-Valverde, M., 2016. Monitoring changes in the structure and properties of humic substances following ozonation using UV\u0026ndash;Vis, FTIR and 1 H NMR techniques. Sci. Total Environ. 541, 623\u0026ndash;637. https://doi.org/10.1016/j.scitotenv.2015.09.127\u003c/li\u003e\n\u003cli\u003eRoscetto, E., Contursi, P., Vollaro, A., Fusco, S., Notomista, E., Catania, M.R., 2018. Antifungal and anti-biofilm activity of the first cryptic antimicrobial peptide from an archaeal protein against Candida spp. clinical isolates. Sci. Rep. 8, 17570. https://doi.org/10.1038/s41598-018-35530-0\u003c/li\u003e\n\u003cli\u003eSaab, S. da C., Carvalho, E.R., Bernardes Filho, R., Moura, M.R. de, Martin-Neto, L., Mattoso, L.H.C., 2010. pH effect in aquatic fulvic acid from a Brazilian river. J. Braz. Chem. Soc. 21, 1490\u0026ndash;1496. https://doi.org/10.1590/S0103-50532010000800012\u003c/li\u003e\n\u003cli\u003eSchnitzer, M., Preston, C.M., 1986. Analysis of humic acids by solution and solid‐state carbon‐13 nuclear magnetic resonance. Soil Sci. Soc. Am. J. 50, 326\u0026ndash;331. https://doi.org/10.2136/sssaj1986.03615995005000020015x\u003c/li\u003e\n\u003cli\u003eSkoog, D.A., Holler, F.J., Crouch, S.R., 1985. Principles of instrumental analysis. Saunders Coll. Publ. USA 344\u0026ndash;347.\u003c/li\u003e\n\u003cli\u003eStevenson, F.J., 1995. Humus chemistry: Genesis, composition, reactions., Second Edi. ed, Journal of Chemical Education. https://doi.org/10.1021/ed072pA93.6\u003c/li\u003e\n\u003cli\u003eTan, K.H., 2014. Humic matter in soil and the environment. CRC Press. https://doi.org/10.1201/b17037\u003c/li\u003e\n\u003cli\u003eTatzber, M., Stemmer, M., Spiegel, H., Katzlberger, C., Zehetner, F., Haberhauer, G., Garcia-Garcia, E., Gerzabek, M.H., 2009. Spectroscopic behaviour of 14C-labeled humic acids in a long-term field experiment with three cropping systems. Soil Res. 47, 459. https://doi.org/10.1071/SR08231\u003c/li\u003e\n\u003cli\u003eWarman, P.R., AngLopez, M.J., 2010. Vermicompost derived from different feedstocks as a plant growth medium. Bioresour. Technol. 101, 4479\u0026ndash;4483. https://doi.org/10.1016/j.biortech.2010.01.098\u003c/li\u003e\n\u003cli\u003eWatanabe, K., Manefield, M., Lee, M., Kouzuma, A., 2009. Electron shuttles in biotechnology. Curr. Opin. Biotechnol. 20, 633\u0026ndash;641. https://doi.org/10.1016/j.copbio.2009.09.006\u003c/li\u003e\n\u003cli\u003eXudan, X., 1986. The effect of foliar application of fulvic acid on water use, nutrient uptake and yield in wheat. Aust. J. Agric. Res. 37, 343. https://doi.org/10.1071/AR9860343\u003c/li\u003e\n\u003cli\u003eYamauchi, N., Toyodome, W., Umeda, K., Nishida, N., Murae, T., 2004. Structural features of humic acid of the coastal sediment in Ariake sea tidelands: Use of humic acid as an environmental indicator for river basins and coastal regions. Anal. Sci. 20, 1453\u0026ndash;1457. https://doi.org/10.2116/analsci.20.1453\u003c/li\u003e\n\u003cli\u003eYang, C.-M., Wang, M.-C., Lu, Y.-F., Chang, I.-F., Chou, C.-H., 2004. Humic substances affect the activity of chlorophyllase. J. Chem. Ecol. 30, 1057\u0026ndash;1065. https://doi.org/10.1023/B:JOEC.0000028467.82191.f9\u003c/li\u003e\n\u003cli\u003eZara, L.F., Rosa, A.H., Toscano, I.A.S., Rocha, J.C., 2006. A structural conformation study of aquatic humic acid. J. Braz. Chem. Soc. 17, 1014\u0026ndash;1019. https://doi.org/10.1590/S0103-50532006000500028\u003c/li\u003e\n\u003cli\u003eZhao, H., Liu, Y., Cui, Z., Beattie, D., Gu, Y., Wang, Q., 2011. Design, synthesis, and biological activities of arylmethylamine substituted chlorotriazine and methylthiotriazine compounds. J. Agric. Food Chem. 59, 11711\u0026ndash;11717. https://doi.org/10.1021/jf203383s\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Humic acid, Fulvic acid, Characterization, Candida, Perionyx excavatus","lastPublishedDoi":"10.21203/rs.3.rs-4221685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4221685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHumic substances are widespread in the world and are formed from the plant and animal leftovers in the habitat when they are decomposed. The particular stuffs include three major combinations like humin, humic and fulvic acid. There are absolutely some reports available regarding the fungicidal activity of humic constituents. The objective of this study was humic materials determination and isolation from organic wastes and vermicompost deroved using the earthworm \u003cem\u003ePerionyx excavatus\u003c/em\u003e for use to strength about the antifungal activity against the human fungal pathogen \u003cem\u003eCandida\u003c/em\u003e species. Characterization of the fulvic and humic substances were done by various methods, including, UV-Vis spectroscopy, Fourier transform infrared, scanning electron microscopy and x-ray diffraction. According to the characterization results, it was observed that humic acid has a structure of honeycomb with fragments and flakes attached to the surface on it. The fractions also have a distinctive physical appearance of lumpy and close-grained. SEM and XRD analysis revealed that certain crystal forms occur in all humic and fulvic acid samples. The absorbances of peak between 250\u0026ndash;500 nm have attributed significance nature of the humic acid. Further, total hydroxyls, amines, alkyl, alcoholic contents and acidic functional groups were determined. The growth of fungal \u003cem\u003eCandida\u003c/em\u003e species was mainly affected by humic and fulvic acid and can reduce the usage of chemical drug that has several side effects. The earthworm plays major role in breakdown of organic substances to form humic substances, which is present in the vermicompost, vermicast and vermiwash. So, the approach of humic and fulvic acid taken from the vermiproduct is used to treat against the human fungal pathogen \u003cem\u003eCandida\u003c/em\u003e species.\u003c/p\u003e","manuscriptTitle":"Isolation, Characterization and Antifungal Behavior of Humic Acid and Fulvic Acid Fractions from Biowaste Derived Vermiproducts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 18:29:51","doi":"10.21203/rs.3.rs-4221685/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24f96c35-5a1d-4def-aacb-995b11d9c7e5","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-10T18:29:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 18:29:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4221685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4221685","identity":"rs-4221685","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}